Journal of Molecular Biology

Conformational dynamics of the Escherichia coli DNA polymerase manager proteins UmuD and UmuD'

Publication Date: 2010 Mar 3 PMID: 20206636
Authors: Fang, J. - Rand, K. D. - Silva, M. C. - Wales, T. E. - Engen, J. R. - Beuning, P. J.
Journal: J Mol Biol

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino acid protein, which subsequently cleaves off its N-terminal 24-amino acids in a RecA/ssDNA-dependent reaction, giving UmuD'. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA pol IV-dependent-1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA pol V (UmuD'(2)C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, has been identified that is noncleavable but is a partial biological mimic of the cleaved form, UmuD'. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD', and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent-accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD', is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD' and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD' because in both cases the arms are relatively unbound from the globular domain. Gas phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter time scales than solution experiments, show that UmuD' incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is likely of critical importance for their many protein interactions.

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Cysteine as a modulator residue in the active site of Xenobiotic Reductase A: A structural, thermodynamical and kinetic study.

Publication Date: 2010 Mar 2 PMID: 20206186
Authors: Spiegelhauer, O. - Mende, S. - Dickert, F. - Knauer, S. H. - Ullmann, G. M. - Dobbek, H.
Journal: J Mol Biol

Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NADH/NADPH-dependent reduction of various substrates, including 2-cyclohexenone and 8-hydroxycoumarin. XenA is a member of the old-yellow-enzyme family of flavoproteins and is structurally and functionally similar to other bacterial members of this enzyme class. A characteristic feature of XenA is the presence of a cysteine residue (Cys25) in the active site, where in most members of the old-yellow-enzyme family a threonine residue is found that modulates the reduction potential of the FMN/FMNH(-)couple. We investigated the role of Cys25 by studying two variants in which the residue has been exchanged for a serine and an alanine residue. While the exchange against alanine has a remarkably small effect on the reduction potential, the reactivity and the structure of XenA, the exchange against serine increases the reduction potential by+82 mV, increases the rate constant of the reductive half-reaction and decreases the rate constant in the oxidative half-reaction. We determined six crystal structures at high to true atomic resolution (d(min): 1.03-1.80 A) of the three XenA variants with and without the substrate coumarin bound in the active site. The atomic resolution structure of XenA in complex with coumarin reveals a compressed active site geometry in which the isoalloxazine ring is sandwiched between coumarin and the protein backbone. The structures further reveal that the conformation of the active site and substrate interactions are preserved in the two variants, indicating that the observed changes are due to local effects only. We propose that Cys25 and the residues in its place determine, depending on the substrate couple, which of the two half-reactions is rate limiting. This may help to explain why the genome of P. putida encodes multiple xenobiotic reductases containing either a cysteine, a threonine or an alanine in the active site.

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Molecular structure of the N-terminal domain of the APC/C subunit Cdc27 reveals a homo-dimeric tetratricopeptide repeat architecture.

Publication Date: 2010 Mar 2 PMID: 20206185
Authors: Zhang, Z. - Roe, S. M. - Diogon, M. - Kong, E. - El Alaoui, H. - Barford, D.
Journal: J Mol Biol

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that targets specific cell cycle regulatory proteins for ubiquitin-dependent degradation, thereby controlling cell cycle events such as the metaphase to anaphase transition and the exit from mitosis. Biochemical and genetic studies are consistent with the notion that subunits of the APC/C are organised into two distinct sub-complexes; a catalytic sub-complex including the cullin domain and RING finger subunits Apc2 and Apc11 respectively, and a tetratricopeptide repeat (TPR) sub-complex composed of the TPR subunits Cdc16, Cdc23 and Cdc27 (Apc3). Here, we describe the crystal structure of the N-terminal domain of E. cuniculi Cdc27 (Cdc27(Nterm)), revealing a homo-dimeric structure, predominantly composed of successive TPR motifs. Mutation of the Cdc27(Nterm) dimer interface destabilises the protein, disrupts dimerisation in solution, and abolishes the capacity of E. cuniculi Cdc27 to complement S. cerevisiae Cdc27 in vivo. These results establish the existence of functional APC/C genes in E. cuniculi, the evolutionarily conserved dimeric properties of Cdc27, and provide a framework for understanding the architecture of full length Cdc27.

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Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis.

Publication Date: 2010 Mar 2 PMID: 20206184
Authors: Fedarovich, A. - Nicholas, R. A. - Davies, C.
Journal: J Mol Biol

PBPA from Mycobacterium tuberculosis (Mtb) is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in Mtb, but is important for proper cell division in M. smegmatis. We have determined the crystal structure of PBPA at 2.05 A resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to Class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active site motifs that characterize penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulphide bridge between Cys282 (the "x" of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between beta5 and alpha11, which restricts the space available in the active site of PBPA, and suggests that conformational changes would be required to accommodate peptide substrate or beta-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.

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Substrate Binding and Catalysis in Carbamate Kinase Ascertained by Crystallographic and Site-Directed Mutagenesis Studies: Movements and Significance of a Unique Globular Subdomain of This Key Enzyme for Fermentative ATP Production in Bacteria.

Publication Date: 2010 Feb 25 PMID: 20188742
Authors: Ramon-Maiques, S. - Marina, A. - Guinot, A. - Gil-Ortiz, F. - Uriarte, M. - Fita, I. - Rubio, V.
Journal: J Mol Biol

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1. 5 A resolution and containing bound MgADP, and the other at 2.1 A resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K(m)(CP) and k(cat) triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.

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Dynamic Elements at Both Cytoplasmically and Extracellularly Facing Sides of the UapA Transporter Selectively Control the Accessibility of Substrates to Their Translocation Pathway.

Publication Date: 2010 Feb 25 PMID: 20188741
Authors: Kosti, V. - Papageorgiou, I. - Diallinas, G.
Journal: J Mol Biol

In the UapA uric acid-xanthine permease of Aspergillusnidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (DeltaazgA DeltafcyB DeltauapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.

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Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase.

Publication Date: 2010 Feb 25 PMID: 20188740
Authors: Scoble, J. - McAlister, A. D. - Fulton, Z. - Troy, S. - Byres, E. - Vivian, J. P. - Brammananth, R. - Wilce, M. C. - Le Nours, J. - Zaker-Tabrizi, L. - Coppel, R. L. - Crellin, P. K. - Rossjohn, J. - Beddoe, T.
Journal: J Mol Biol

Aldo-keto reductases (AKRs) are a large superfamily of NADPH-dependent enzymes that catalyze the reduction of aldehydes, aldoses, dicarbonyls, steroids, and monosaccharides. While their precise physiological role is generally unknown, AKRs are nevertheless involved in the detoxification of a broad range of toxic metabolites. Mycobacteria contain a number of AKRs, the majority of which are uncharacterised. Here, we report the 1.9 and 1.6 A resolution structures of the apoenzyme and NADPH-bound forms, respectively, of an AKR (MSMEG_2407) from Mycobacterium smegmatis, a close homologue of the M. tuberculosis enzyme Rv2971, whose function is essential to this bacterium. MSMEG_2407 adopted the triosephosphate isomerase (alpha/beta)(8)-barrel fold exhibited by other AKRs. MSMEG_2407 (AKR5H1) bound NADPH via an induced-fit mechanism, in which the NADPH was ligated in an extended fashion. Polar-mediated interactions dominated the interactions with the cofactor, which is atypical of the mode of NADPH binding within the AKR family. Moreover, the nicotinamide ring of NADPH was disordered, and this was attributed to the lack of an "AKR-conserved" bulky residue within the nicotinamide-binding cavity of MSMEG_2407. Enzymatic characterisation of MSMEG_2407 and Rv2971 identified dicarbonyls as a preferred substrate family for hydrolysis, and the frontline antituberculosis drug isoniazid (INH) was shown to inhibit the enzyme activity of both recombinant MSMEG_2407 and Rv2971. However, differences between the affinities of MSMEG_2407 and Rv2971 for dicarbonyls and INH were observed, and this was attributable to amino acid substitutions within the cofactor- and substrate-binding sites. The structures of MSMEG_2407 and the accompanying biochemical characterisation of MSMEG_2407 and Rv2971 provide insight into the structure and function of AKRs from mycobacteria.

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Inhibition of Mammary Carcinoma Cell Growth by RXR is Mediated by the Receptor's Oligomeric Switch.

Publication Date: 2010 Feb 24 PMID: 20188110
Authors: Yasmin, R. - Kannan-Thulasiraman, P. - Kagechika, H. - Dawson, M. I. - Noy, N.
Journal: J Mol Biol

Ligands that activate the nuclear receptor retinoid X receptor (RXR) display potent anticarcinogenic activities, but the mechanisms by which these compounds inhibit carcinoma cell growth are poorly understood. While RXR can regulate gene expression due to its intrinsic ligand-activated transcription function, this receptor can also regulate transcription by functioning as a ligand-controlled DNA architectural factor. It was thus reported that apo-RXR self-associates into tetramers and that each dimer within these tetramers can separately bind to an RXR response element. Hence, DNA binding by RXR tetramers may bring distant genomic regions into close physical proximity. As ligand binding induces the dissociation of RXR tetramers into dimers, it can alter gene expression by modulating the DNA architecture. Here, we show that inhibition of mammary carcinoma cell growth by RXR ligands stems from the ability of these compounds to regulate the oligomeric state of RXR and is independent of the direct intrinsic transcriptional activity of the receptor. The data suggest that compounds that trigger dissociation of RXR tetramers may comprise a novel class of anticarcinogenic agents.

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The Effect of Ribosome Assembly Cofactors on In Vitro 30S Subunit Reconstitution.

Publication Date: 2010 Feb 24 PMID: 20188109
Authors: Bunner, A. E. - Nord, S. - Wikstrom, P. M. - Williamson, J. R.
Journal: J Mol Biol

Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Escherichia coli Ras-like protein caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. Ribosome maturation factor M enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. Ribosome maturation factor P caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis.

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Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion.

Publication Date: 2010 Feb 23 PMID: 20188108
Authors: Baird, N. J. - Gong, H. - Zaheer, S. S. - Freed, K. F. - Pan, T. - Sosnick, T. R.
Journal: J Mol Biol

RNA folding occurs via a series of transitions between metastable intermediate states for Mg(2+) concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.

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Structural and Biochemical Bases for the Redox Sensitivity of Mycobacterium tuberculosis RslA.

Publication Date: 2010 Feb 22 PMID: 20184899
Authors: Thakur, K. G. - Praveena, T. - Gopal, B.
Journal: J Mol Biol

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis sigma factor sigma(L) regulates the expression of genes involved in cell-wall and polyketide syntheses. sigma(L) interacts with the cytosolic anti-sigma domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn(2+) and can sequester sigma(L) in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn(2+). This results in a substantial rearrangement of the sigma(L)/RslA complex, leading to an 8-fold decrease in the affinity of RslA for sigma(L). The crystal structure of the -35-element recognition domain of sigma(L), sigma(4)(L), bound to RslA reveals that RslA inactivates sigma(L) by sterically occluding promoter DNA and RNA polymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn(2+) in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of sigma(L)/RslA interactions provide a template for understanding how variations in the rate of Zn(2+) release and associated conformational changes could regulate the activity of a Zn(2+)-associated anti-sigma factor.

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Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity.

Publication Date: 2010 Feb 23 PMID: 20184898
Authors: Nesmelova, I. V. - Ermakova, E. - Daragan, V. A. - Pang, M. - Menendez, M. - Lagartera, L. - Solis, D. - Baum, L. G. - Mayo, K. H.
Journal: J Mol Biol

Galectins are a subfamily of lectins with a conserved carbohydrate recognition domain that interacts with beta-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the beta-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K(1)=21+/-6x10(3) M(-1)) than the second (K(2)=4+/-2x10(3) M(-1)). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K(1)=20+/-10x10(3) M(-1) and K(2)=1.67+/-0.07x10(3) M(-1). Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the beta-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.

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Crystal Structure of Prostate Secretory Protein PSP94 Shows an Edge-to-Edge Association of two Monomers to Form a Homodimer.

Publication Date: 2010 Feb 23 PMID: 20184897
Authors: Kumar, A. - Jagtap, D. D. - Mahale, S. D. - Kumar, M.
Journal: J Mol Biol

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 A resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last beta-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.

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Conversion of a Regulatory into a Degradative Protease.

Publication Date: 2010 Feb 22 PMID: 20184896
Authors: Hasenbein, S. - Meltzer, M. - Hauske, P. - Kaiser, M. - Huber, R. - Clausen, T. - Ehrmann, M.
Journal: J Mol Biol

The PDZ protease DegS senses mislocalized outer membrane proteins and initiates the sigmaE pathway in the bacterial periplasm. This unfolded protein response pathway is activated by processing of the anti-sigma factor RseA by DegS and other proteases acting downstream of DegS. DegS mediates the rate-limiting step of sigma E induction and its activity must be highly specific and tightly regulated. While DegS is structurally and biochemically well studied, the determinants of its pronounced substrate specificity are unknown. We therefore performed swapping experiments by introducing elements of the homologous but unspecific PDZ protease DegP. Introduction of loop L2 of DegP into DegS converted the enzyme into a non-specific protease, while swapping of PDZ domains did not. Therefore, loop L2 of the protease domain is a key determinant of substrate specificity. Interestingly, swapping of loop L2 did not affect the tight regulation of DegS. In addition, the combined introduction of loop L2 and PDZ domain 1 of DegP into DegS converted DegS even further into a DegP-like protease. These and other data suggest that homologous enzymes with distinct activities and regulatory features can be converted by simple genetic modifications.

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The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor.

Publication Date: 2010 Feb 22 PMID: 20184895
Authors: Sankaranarayanan, R. - Cherney, M. M. - Garen, C. - Garen, G. - Niu, C. - Yuan, M. - James, M. N.
Journal: J Mol Biol

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 A resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O(gamma1) of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.

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Comprehensive Analysis of HAMP Domains: Implications for Transmembrane Signal Transduction.

Publication Date: 2010 Feb 23 PMID: 20184894
Authors: Dunin-Horkawicz, S. - Lupas, A. N.
Journal: J Mol Biol

Homodimeric receptors with one or two transmembrane (TM) segments per monomer are universal to life and represent the largest and most diverse group of cellular TM receptors. They frequently share domain types across phyla and, in some cases, have been recombined experimentally into functional chimeras (e.g., the bacterial aspartate chemoreceptor with the human insulin receptor), suggesting that they have a common mechanism. The nature of this mechanism, however, is still being debated. We have proposed a new model for transduction mechanism by axial helix rotation, based on the structure of a widespread domain, HAMP, that frequently occurs in direct continuation of the last TM segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of coevolving residues, which allow the mechanism to subdivide into individual steps. The most extended of these networks is specific for membrane-bound HAMP domains and most likely accepts the signal from the TM helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classification shows a division between domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive coevolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts.

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Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species.

Publication Date: 2010 Feb 23 PMID: 20184893
Authors: Kayatekin, C. - Zitzewitz, J. A. - Matthews, C. R.
Journal: J Mol Biol

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral pH and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to approximately 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the stable native dimeric state.

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The Membrane Complex between Transducin and Dark-State Rhodopsin Exhibits Large-Amplitude Interface Dynamics on the Sub-Microsecond Timescale: Insights from All-Atom MD Simulations.

Publication Date: 2010 Feb 23 PMID: 20184892
Authors: Sgourakis, N. G. - Garcia, A. E.
Journal: J Mol Biol

Rhodopsin, the prototype class A G-protein-coupled receptor, is a very important model system for all seven-transmembrane domain proteins. Characterization of the interactions between rhodopsin and transducin, its intracellular G-protein counterpart, and the fluctuations in these interactions due to thermal motions is required for an understanding of early events in the mechanism of signal transduction. In this study, we used all-atom molecular dynamics simulations of a transmembrane protein complex between rhodopsin and the heterotrimeric transducin (Galphabetagamma) in an all-atom DOPC (1,2-dioleoylsn-glycero-3-phosphocholine) membrane-water environment. Based on the analysis of a microsecond-timescale simulation trajectory, we characterized the dynamics of the system and its effects in the structural features of the protein subunits. Our simulations describe a highly dynamic interaction interface where the system is alternating between distinct domain orientations at the 10- to 100-ns timescale that can be further classified into interaction modes involving contacts between distinct structural features on the protein subunits. We related our results with experimental measurements from a variety of studies and high-resolution models of activated rhodopsin. Monitoring key structural features that are involved in the activation process along our simulation trajectory indicates the presence of extensive dynamics in the dark-adapted state, including a motion of Y223 from helix 3 toward the "ionic-lock" interactions of the conserved ERY motif. The dynamic picture shown here is consistent with a framework in which the dark-state fluctuations sample conformations consistent with the activated state. These results provide an atomic-level description of the dynamics of the full complex and further suggest novel mutagenesis experiments that can be used to investigate the stability and dynamics of this model membrane protein receptor system.

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Crystal Structure of P58(IPK) TPR Fragment Reveals the Mechanism for its Molecular Chaperone Activity in UPR.

Publication Date: 2010 Feb 22 PMID: 20184891
Authors: Tao, J. - Petrova, K. - Ron, D. - Sha, B.
Journal: J Mol Biol

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 A resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).

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Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid.

Publication Date: 2010 Feb 23 PMID: 20184890
Authors: Pohjoismaki, J. L. - Holmes, J. B. - Wood, S. R. - Yang, M. Y. - Yasukawa, T. - Reyes, A. - Bailey, L. J. - Cluett, T. J. - Goffart, S. - Willcox, S. - Rigby, R. E. - Jackson, A. P. - Spelbrink, J. N. - Griffith, J. D. - Crouch, R. J. - Jacobs, H. T. - Holt, I. J.
Journal: J Mol Biol

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.

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Regions Outside the alpha-Crystallin Domain of the Small Heat Shock Protein Hsp26 Are Required for Its Dimerization.

Publication Date: 2010 Feb 18 PMID: 20171228
Authors: Chen, J. - Feige, M. J. - Franzmann, T. M. - Bepperling, A. - Buchner, J.
Journal: J Mol Biol

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones. They form homo-oligomers, composed of mostly 24 subunits. The immunoglobulin-like alpha-crystallin domain, which is flanked by N- and C-terminal extensions, is the most conserved element in sHsps. It is assumed to be the dimeric building block from which the sHsp oligomers are assembled. Hsp26 from Saccharomyces cerevisiae is a well-characterized member of this family. With a view to study the structural stability and oligomerization properties of its alpha-crystallin domain, we produced a series of alpha-crystallin domain constructs. We show that a minimal alpha-crystallin domain can, against common belief, be monomeric and stably folded. Elongating either the N- or the C-terminus of this minimal alpha-crystallin domain with the authentic extensions leads to the formation of dimeric species. In the case of N-terminal extensions, their population is dependent on the presence of the complete so-called Hsp26 "middle domain". For the C-terminal extensions, the presence of the conserved IXI motif of sHsps is necessary and sufficient to induce dimerization, which can be inhibited by increasing ionic strength. Dimerization does not induce major changes in secondary structure of the Hsp26 alpha-crystallin domain. A thermodynamic analysis of the monomeric and dimeric constructs revealed that dimers are not significantly stabilized against thermal and chemical denaturation in comparison to monomers, supporting our notion that dimerization is not a prerequisite for the formation of a well-folded Hsp26 alpha-crystallin domain.

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pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the beta-Barrel Membrane Protein OmpG.

Publication Date: 2010 Feb 18 PMID: 20171227
Authors: Damaghi, M. - Bippes, C. - Koster, S. - Yildiz, O. - Mari, S. A. - Kuhlbrandt, W. - Muller, D. J.
Journal: J Mol Biol

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the beta-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of beta-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.

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Mutations in Desmin's Carboxy-Terminal "Tail" Domain Severely Modify Filament and Network Mechanics.

Publication Date: 2010 Feb 18 PMID: 20171226
Authors: Bar, H. - Schopferer, M. - Sharma, S. - Hochstein, B. - Mucke, N. - Herrmann, H. - Willenbacher, N.
Journal: J Mol Biol

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-alpha-helical carboxy-terminal domain ("tail"), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin "tail" mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l(p). Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the "tail" mutants (l(p)=1.0-2.0 mum) and the wild-type desmin (l(p)=1.1+/-0.5 mum). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered. In order to explore the importance of the "tail" domain as such for filament network properties, we employed a "tail"-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the "tail" domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.

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Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure.

Publication Date: 2010 Feb 12 PMID: 20156459
Authors: Andersen, C. B. - Hicks, M. R. - Vetri, V. - Vandahl, B. - Rahbek-Nielsen, H. - Thogersen, H. - Thogersen, I. B. - Enghild, J. J. - Serpell, L. C. - Rischel, C. - Otzen, D. E.
Journal: J Mol Biol

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional beta-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from beta-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.

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Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry.

Publication Date: 2010 Feb 12 PMID: 20156458
Authors: Baum, B. - Muley, L. - Smolinski, M. - Heine, A. - Hangauer, D. - Klebe, G.
Journal: J Mol Biol

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.

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The Subunit Interfaces of Weakly Associated Homodimeric Proteins.

Publication Date: 2010 Feb 13 PMID: 20156457
Authors: Dey, S. - Pal, A. - Chakrabarti, P. - Janin, J.
Journal: J Mol Biol

We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

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HBx-Induced Hepatic Steatosis and Apoptosis Are Regulated by TNFR1- and NF-kappaB-Dependent Pathways.

Publication Date: 2010 Feb 13 PMID: 20156456
Authors: Kim, J. Y. - Song, E. H. - Lee, H. J. - Oh, Y. K. - Choi, K. H. - Yu, D. Y. - Park, S. I. - Seong, J. K. - Kim, W. H.
Journal: J Mol Biol

Hepatitis B virus X (HBx) protein is an important regulator of hepatic steatosis observed in patients with hepatitis B virus; however, its underlying molecular mechanism remains unclear. TNF receptor 1 (TNFR1) is an essential pathway for the HBx-mediated nuclear factor kappaB (NF-kappaB) activation involved in hepatic liver injury. Here, we show that HBx-mediated steatosis and apoptosis are regulated by TNFR1- and NF-kappaB-dependent pathways. HBx-mediated tumor necrosis factor alpha (TNF-alpha) production and NF-kappaB activation were completely diminished in anti-TNF-alpha-treated cells and TNF-alpha(-)(/-) or TNFR1(-/-) mice. HBx and TNFR1, which are potentiated by TNF-alpha, are physically associated and colocalize in the plasma membrane. Similarly, TNFR1 depletion inhibits lipid droplets, and lipogenic genes such as sterol regulatory element binding protein (SREBP) 1 and peroxisome proliferator-activated receptor (PPAR) gamma increased in HBx-Tg mice and HepG2-GFPHBx stable cells. Furthermore, lipid accumulation and expression of SREBP1c and PPARgamma are significantly increased in AdHBx-GFP-injected (intravenous) wild-type mice, but not in TNFR1(-/-) mice. HBx-enhanced transcriptional activities of SREBP1 and PPARgamma are significantly attenuated by the NF-kappaB inhibitor Bay 11-7082, as well as by TNFR1 depletion. Also, AdHBx-GFP potentiates TNF-alpha-induced apoptosis, which is completely inhibited in TNFR1-depleted cells. Our results suggest that HBx-induced NF-kappaB activation was mediated by direct interaction with TNFR1 and thereby induced TNF-alpha production. HBx-induced NF-kappaB activation is also associated with the induction of hepatic steatosis and apoptosis, which is determined by a TNFR1-dependent pathway.

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The Asymmetric Flagellar Distribution and Motility of Escherichia coli.

Publication Date: 2010 Feb 13 PMID: 20156455
Authors: Ping, L.
Journal: J Mol Biol

Rod-shaped bacteria such as Escherichia coli divide by binary fission. They inherit an old pole from the parent cell. The new pole is recently derived from the septum. Because the chemoreceptor accumulates linearly with time on the cell pole, the old pole carries more receptors than does the new pole. Here, further evidence is provided that the old pole appears more frequently at the rear when bacteria swim. This phenomenon had been observed, yet not extensively explored in the literature. The biased swimming orientation is the consequence of the asymmetric distribution of flagella over the cell surface. On about 75% of cells, there are more flagella on the old-pole half of the cell than on the new-pole half, regardless of growth conditions. Most flagella are lateral, and few were found on the cell pole per se. The asymmetric flagellar distribution makes cells more efficient in chemotaxis. Both swimming orientation and receptor localization are components of chemotaxis, by which bacteria follow environmental stimuli. If unipolarly flagellated cells, such as the swarmer cells of Caulobacter crescentus, are regarded as 100% polar with respect to chemotaxis, E. coli is about 75%. The difference is quantitative. The peritrichous flagellation might enhance the motility and chemotaxis in the viscous environment of enteric bacteria.

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Visualizing the Molecular Interactions of a Nucleotide Analog, GS-9148, with HIV-1 Reverse Transcriptase-DNA Complex.

Publication Date: 2010 Feb 13 PMID: 20156454
Authors: Lansdon, E. B. - Samuel, D. - Lagpacan, L. - Brendza, K. M. - White, K. L. - Hung, M. - Liu, X. - Boojamra, C. G. - Mackman, R. L. - Cihlar, T. - Ray, A. S. - McGrath, M. E. - Swaminathan, S.
Journal: J Mol Biol

GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phospho nic acid) is a dAMP (2'-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2'-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT-DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 A shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable pi-pi interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2'-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.

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Structure of Bacteriophage T4 Endonuclease II Mutant E118A, a Tetrameric GIY-YIG Enzyme.

Publication Date: 2010 Feb 13 PMID: 20156453
Authors: Andersson, C. E. - Lagerback, P. - Carlson, K.
Journal: J Mol Biol

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2(1) with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.

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Structures of PHR Domains from Mus musculus Phr1 (Mycbp2) Explain the Loss-of-Function Mutation (Gly1092-->Glu) of the C. elegans Ortholog RPM-1.

Publication Date: 2010 Feb 13 PMID: 20156452
Authors: Sampathkumar, P. - Ozyurt, S. A. - Miller, S. - Bain, K. T. - Rutter, M. E. - Gheyi, T. - Abrams, B. - Wang, Y. - Atwell, S. - Luz, J. G. - Thompson, D. A. - Wasserman, S. R. - Emtage, J. S. - Park, E. C. - Rongo, C. - Jin, Y. - Klemke, R. L. - Sauder, J. M. - Burley, S. K.
Journal: J Mol Biol

PHR [PAM (protein associated with Myc)-Highwire (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel beta sandwich fold composed of 11 antiparallel beta-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092-->Glu, observed in the Caenorhabditis elegans ortholog RPM-1.

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Exit Strategies for Charged tRNA from GluRS.

Publication Date: 2010 Feb 13 PMID: 20156451
Authors: Pyrkosz, A. B. - Eargle, J. - Sethi, A. - Luthey-Schulten, Z.
Journal: J Mol Biol

For several class I aminoacyl-tRNA synthetases (aaRSs), the rate-determining step in aminoacylation is the dissociation of charged tRNA from the enzyme. In this study, the following factors affecting the release of the charged tRNA from aaRSs are computationally explored: the protonation states of amino acids and substrates present in the active site, and the presence and the absence of AMP and elongation factor Tu. Through molecular modeling, internal pK(a) calculations, and molecular dynamics simulations, distinct, mechanistically relevant posttransfer states with charged tRNA bound to glutamyl-tRNA synthetase from Thermus thermophilus (Glu-tRNA(Glu)) are considered. The behavior of these nonequilibrium states is characterized as a function of time using dynamic network analysis, local energetics, and changes in free energies to estimate transitions that occur during the release of the tRNA. The hundreds of nanoseconds of simulation time reveal system characteristics that are consistent with recent experimental studies. Energetic and network results support the previously proposed mechanism in which the transfer of amino acid to tRNA is accompanied by the protonation of AMP to H-AMP. Subsequent migration of proton to water reduces the stability of the complex and loosens the interface both in the presence and in the absence of AMP. The subsequent undocking of AMP or tRNA then proceeds along thermodynamically competitive pathways. Release of the tRNA acceptor stem is further accelerated by the deprotonation of the alpha-ammonium group on the charging amino acid. The proposed general base is Glu41, a residue binding the alpha-ammonium group that is conserved in both structure and sequence across nearly all class I aaRSs. This universal handle is predicted through pK(a) calculations to be part of a proton relay system for destabilizing the bound charging amino acid following aminoacylation. Addition of elongation factor Tu to the aaRS.tRNA complex stimulates the dissociation of the tRNA core and the tRNA acceptor stem.

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Regulation of Aerobic-to-Anaerobic Transitions by the FNR Cycle in Escherichia coli.

Publication Date: 2010 Feb 12 PMID: 20156450
Authors: Tolla, D. A. - Savageau, M. A.
Journal: J Mol Biol

The FNR (fumarate nitrate reduction) protein plays a central role in the global oxygen response of a variety of bacteria. In Escherichia coli, FNR is the master transcriptional regulator of the transition between aerobic and anaerobic growth. Regulation of FNR is achieved by cycling the molecule between three states in a process dependent on oxygen. In an effort to better understand the nature of this post-transcriptional cyclic regulatory mechanism, we formulated a kinetic model of the FNR protein and its regulation in E. coli. The values for the parameters of the model were fit to experimental data for the wild-type organism, and the model was validated by successfully predicting the behavior of fnr mutant strains characterized in the literature. We characterized the steady-state behavior of the FNR system by determining its sensitivity to changes in parameter values and its response to changes in the concentration of iron-sulfur cluster assembly proteins and the protease ClpXP. We also determined the steady-state induction characteristic that provides a direct estimate for the levels of the active form of FNR as a function of oxygen concentration. This result, in combination with reporter assays for expression of FNR target operons, gives an estimate for the equilibrium dissociation constant for the binding of active FNR to its recognition sequences in the DNA. Finally, we predicted the dynamics of the aerobic-to-anaerobic transition and determined distinct contributions to the dynamic profile of regulatory mechanisms operating at the transcriptional and post-translational levels.

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Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA.

Publication Date: 2010 Feb 12 PMID: 20156449
Authors: Popp, D. - Narita, A. - Ghoshdastider, U. - Maeda, K. - Maeda, Y. - Oda, T. - Fujisawa, T. - Onishi, H. - Ito, K. - Robinson, R. C.
Journal: J Mol Biol

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure SAX experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.

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Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities.

Publication Date: 2010 Feb 12 PMID: 20156448
Authors: Szymanski, M. R. - Jezewska, M. J. - Bujalowski, W.
Journal: J Mol Biol

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12+/-1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induced a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.

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Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome c(z) Subunit from Green Photosynthetic Bacterium Chlorobium tepidum.

Publication Date: 2010 Feb 12 PMID: 20156447
Authors: Hirano, Y. - Higuchi, M. - Azai, C. - Oh-Oka, H. - Miki, K. - Wang, Z. Y.
Journal: J Mol Biol

In green sulfur photosynthetic bacteria, the cytochrome c(z) (cyt c(z)) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c(z) subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c(z) subunit (C-cyt c(z)) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-A resolution. The overall fold of C-cyt c(z) consists of four alpha-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c(z) supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c(z).

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Vesicle Permeabilization by Purified Soluble Oligomers of Prion Protein: A Comparative Study of the Interaction of Oligomers and Monomers with Lipid Membranes.

Publication Date: 2010 Feb 13 PMID: 20156446
Authors: Chich, J. F. - Chapuis, C. - Henry, C. - Vidic, J. - Rezaei, H. - Noinville, S.
Journal: J Mol Biol

The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

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Structure of Avian Thymic Hormone, a High-Affinity Avian beta-Parvalbumin, in the Ca(2+)-Free and Ca(2+)-Bound States.

Publication Date: 2010 Feb 12 PMID: 20156445
Authors: Schuermann, J. P. - Tan, A. - Tanner, J. J. - Henzl, M. T.
Journal: J Mol Biol

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two beta-lineage isoforms expressed in birds. We recently learned that addition of Ca(2+)-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca(2+), suggests that apolar surface area buried in the Ca(2+)-bound state becomes solvent accessible upon Ca(2+) removal. In order to elucidate the conformational alterations that accompany Ca(2+) binding, we have obtained the solution structure of the Ca(2+)-free protein using NMR spectroscopy and compared it to the Ca(2+)-loaded protein, solved by X-ray crystallography. Although the metal-ion-binding (CD-EF) domains are largely coincident in the superimposed structures, a major difference is observed in the AB domains. The tight association of helix B with the E and F helices in the Ca(2+)-bound state is lost upon removal of Ca(2+), producing a deep hydrophobic cavity. The B helix also undergoes substantial rotation, exposing the side chains of F24, Y26, F29, and F30 to solvent. Presumably, the increase in ANS emission observed in the presence of unliganded ATH reflects the interaction of these hydrophobic residues with the fluorescent probe. The increased solvent exposure of apolar surface area in the Ca(2+)-free protein is consistent with previously collected scanning calorimetry data, which indicated an unusually low change in heat capacity upon thermal denaturation. The Ca(2+)-free structure also provides added insight into the magnitude of ligation-linked conformational alteration compatible with a high-affinity metal-ion-binding signature. The exposure of substantial apolar surface area suggests the intriguing possibility that ATH could function as a reverse Ca(2+) sensor.

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Solution Structure of Gfi-1 Zinc Domain Bound to Consensus DNA.

Publication Date: 2010 Feb 11 PMID: 20153336
Authors: Lee, S. - Doddapaneni, K. - Hogue, A. - McGhee, L. - Meyers, S. - Wu, Z.
Journal: J Mol Biol

Gfi-1 is a crucial transcriptional repressor for the precise regulation of cell proliferation and differentiation in hematopoiesis. Recently, this protein has also been demonstrated to be capable of restricting the proliferation of hematopoietic stem cells, a process that appears to be vital for the long-term competency of hematopoietic stem cells. These two seemingly opposite outcomes of regulation are likely to arise from its interactions with a variety of cellular partners. Such interactions can directly affect the genes that Gfi-1 recognizes through its DNA binding zinc-finger domain. In this work, we report the determination of the solution structure of Gfi-1 zinc fingers 3-5 in complex with a 16-mer consensus DNA using multidimensional NMR method. Unlike a proposed minor-groove binding model based on methylation interference experiments, our structure clearly shows that Gfi-1 zinc fingers 3-5 bind into the major groove of the target DNA reminiscent of canonical C(2)H(2) zinc-finger domains. The fourth and fifth zinc fingers recognize the AATC core sequence by forming base-specific hydrogen bonds between the side chains of Asn382, Gln379, and Asp354 and the bases of the invariant adenines and cytosine. Overall, the current work provides valuable insight into the structural determinants for DNA binding specificity, in particular for the TCA triplet that has not been observed in any other structures of zinc finger-DNA complexes, as well as molecular rationales for a naturally occurring mutation that causes acute myeloid leukemia.

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Unfinished Stories on Viral Quasispecies and Darwinian Views of Evolution.

Publication Date: 2010 Feb 10 PMID: 20152841
Authors: Mas, A. - Lopez-Galindez, C. - Cacho, I. - Gomez, J. - Martinez, M. A.
Journal: J Mol Biol

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis.

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Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association.

Publication Date: 2010 Feb 10 PMID: 20149799
Authors: Tobin, C. - Mandava, C. S. - Ehrenberg, M. - Andersson, D. I. - Sanyal, S.
Journal: J Mol Biol

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a DeltaS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA(fMet), and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.

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Structural Basis of Human CYP51 Inhibition by Antifungal Azoles.

Publication Date: 2010 Feb 10 PMID: 20149798
Authors: Strushkevich, N. - Usanov, S. A. - Park, H. W.
Journal: J Mol Biol

The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (cytochrome P40, family 51) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B' helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

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Protein Folding Requires Crowd Control in a Simulated Cell.

Publication Date: 2010 Feb 10 PMID: 20149797
Authors: Jefferys, B. R. - Kelley, L. A. - Sternberg, M. J.
Journal: J Mol Biol

Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have computationally studied the effect of crowding upon protein folding for 12 small domains in a simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics, designed to unify the often disjoint goals of protein folding simulation and structure prediction. The model can make predictions of native conformation with accuracy comparable with that of the best current template-free models. It is fast enough to enable a more extensive analysis of crowding than previously attempted, studying several proteins at many crowding levels and further random repetitions designed to more closely approximate the ensemble of conformations. We found that when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in extended conformations. These results suggest that the ability of small protein domains to fold without the help of chaperones may be an important factor in limiting the degree of macromolecular crowding in the cell. Here, we discuss the possible implications regarding the relationship between protein expression level, protein size, chaperone activity and aggregation.

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Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs.

Publication Date: 2010 Mar 26 PMID: 20132829
Authors: Merritt, E. A. - Arakaki, T. L. - Gillespie, J. R. - Larson, E. T. - Kelley, A. - Mueller, N. - Napuli, A. J. - Kim, J. - Zhang, L. - Verlinde, C. L. - Fan, E. - Zucker, F. - Buckner, F. S. - Van Voorhis, W. C. - Hol, W. G.
Journal: J Mol Biol

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

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Structure of the Human Fatty Acid Synthase KS-MAT Didomain as a Framework for Inhibitor Design.

Publication Date: 2010 Mar 26 PMID: 20132826
Authors: Pappenberger, G. - Benz, J. - Gsell, B. - Hennig, M. - Ruf, A. - Stihle, M. - Thoma, R. - Rudolph, M. G.
Journal: J Mol Biol

The human fatty acid synthase (FAS) is a key enzyme in the metabolism of fatty acids and a target for antineoplastic and antiobesity drug development. Due to its size and flexibility, structural studies of mammalian FAS have been limited to individual domains or intermediate-resolution studies of the complete porcine FAS. We describe the high-resolution crystal structure of a large part of human FAS that encompasses the tandem domain of beta-ketoacyl synthase (KS) connected by a linker domain to the malonyltransferase (MAT) domain. Hinge regions that allow for substantial flexibility of the subdomains are defined. The KS domain forms the canonical dimer, and its substrate-binding site geometry differs markedly from that of bacterial homologues but is similar to that of the porcine orthologue. The didomain structure reveals a possible way to generate a small and compact KS domain by omitting a large part of the linker and MAT domains, which could greatly aid in rapid screening of KS inhibitors. In the crystal, the MAT domain exhibits two closed conformations that differ significantly by rigid-body plasticity. This flexibility may be important for catalysis and extends the conformational space previously known for type I FAS and 6-deoxyerythronolide B synthase.

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Structure of the MADS-box/MEF2 Domain of MEF2A Bound to DNA and Its Implication for Myocardin Recruitment.

Publication Date: 2010 Mar 26 PMID: 20132824
Authors: Wu, Y. - Dey, R. - Han, A. - Jayathilaka, N. - Philips, M. - Ye, J. - Chen, L.
Journal: J Mol Biol

Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1-MEF2B-DNA complex and the HDAC9 (histone deacetylase 9)-MEF2B-DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.

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The Structure and Stability of the Monomorphic HLA-G Are Influenced by the Nature of the Bound Peptide.

Publication Date: 2010 Mar 26 PMID: 20122941
Authors: Walpole, N. G. - Kjer-Nielsen, L. - Kostenko, L. - McCluskey, J. - Brooks, A. G. - Rossjohn, J. - Clements, C. S.
Journal: J Mol Biol

The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse alphabeta T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-G(KGPPAALTL) was very similar to that of the HLA-G(RIIPRHLQL) structure. However, the structure of HLA-G(KLPQAFYIL) not only differed in the conformation of the bound peptide but also caused a small shift in the alpha2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.

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X-ray Crystal Structure of the Rotavirus Inner Capsid Particle at 3.8 A Resolution.

Publication Date: 2010 Mar 26 PMID: 20122940
Authors: McClain, B. - Settembre, E. - Temple, B. R. - Bellamy, A. R. - Harrison, S. C.
Journal: J Mol Biol

The rotavirus inner capsid particle, known as the "double-layered particle" (DLP), is the "payload" delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter approximately 700 A) in the asymmetric unit of the P2(1)2(1)2(1) unit cell of dimensions a=740 A, b=1198 A, and c=1345 A. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 A resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 A). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two "subdomain" boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: lambda1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a "5-fold hub" that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5'-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.

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Multiple Nucleotide Preferences Determine Cleavage-Site Recognition by the HIV-1 and M-MuLV RNases H.

Publication Date: 2010 Mar 19 PMID: 20122939
Authors: Schultz, S. J. - Zhang, M. - Champoux, J. J.
Journal: J Mol Biol

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design.

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DNA packaging-associated hyper-capsid expansion of bacteriophage t3.

Publication Date: 2010 Mar 26 PMID: 20122936
Authors: Serwer, P. - Wright, E. T. - Hakala, K. - Weintraub, S. T. - Su, M. - Jiang, W.
Journal: J Mol Biol

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A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline.

Publication Date: 2010 Mar 26 PMID: 20122935
Authors: Biswas, T. - Zolova, O. E. - Lombo, F. - de la Calle, F. - Salas, J. A. - Tsodikov, O. V. - Garneau-Tsodikova, S.
Journal: J Mol Biol

Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 A resolution reveals that TioX protomer shares an ancient betaalphabetabetabeta fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion.

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Structural Basis for Asymmetric Association of the betaPIX Coiled Coil and Shank PDZ.

Publication Date: 2010 Mar 26 PMID: 20117114
Authors: Im, Y. J. - Kang, G. B. - Lee, J. H. - Park, K. R. - Song, H. E. - Kim, E. - Song, W. K. - Park, D. - Eom, S. H.
Journal: J Mol Biol

betaPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of betaPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of betaPIX, which is important for its physiological functions. We have solved the crystal structure of the betaPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The betaPIX CC forms a 76-A-long parallel CC trimer. Despite the fact that the betaPIX CC exposes three PDZ binding motifs in the C-termini, the betaPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive beta-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of betaPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the betaPIX trimer. The structure shows canonical class I PDZ binding with a beta-sheet interaction extending to position -6 of betaPIX. The betaB-betaC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the beta-sheet interaction and to accommodate the bulky side chain of Trp -5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of betaPIX CC and Shank PDZ.

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A Tag at the Carboxy Terminus Prevents Membrane Integration of VDAC1 in Mammalian Mitochondria.

Publication Date: 2010 Mar 19 PMID: 20117113
Authors: Kozjak-Pavlovic, V. - Ross, K. - Gotz, M. - Goosmann, C. - Rudel, T.
Journal: J Mol Biol

beta-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial beta-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting beta-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of beta-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the beta-signal is shortened, removed or when the conserved hydrophobic residues in the beta-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the beta-signal or the folding of the beta-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of beta-barrel proteins, but also advises caution when interpreting the outcome of the beta-barrel protein overexpression experiments.

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Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles.

Publication Date: 2010 Mar 26 PMID: 20114054
Authors: Roman, E. A. - Arguello, J. M. - Gonzalez Flecha, F. L.
Journal: J Mol Biol

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is involved in transporting Cu(+) throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.

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Frameshifting in Alphaviruses: A Diversity of 3' Stimulatory Structures.

Publication Date: 2010 Mar 26 PMID: 20114053
Authors: Chung, B. Y. - Firth, A. E. - Atkins, J. F.
Journal: J Mol Biol

Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal -1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the -1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein. The present work shows that there is remarkable diversity in the 3' sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3' RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3'-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of -1 frameshifting stimulators in mammalian and insect systems.

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Altered Ion Channel Formation by the Parkinson's-Disease-Linked E46K Mutant of alpha-Synuclein Is Corrected by GM3 but Not by GM1 Gangliosides.

Publication Date: 2010 Mar 19 PMID: 20114052
Authors: Di Pasquale, E. - Fantini, J. - Chahinian, H. - Maresca, M. - Taieb, N. - Yahi, N.
Journal: J Mol Biol

alpha-Synuclein (alpha-syn) is an amyloidogenic protein that plays a key role in the pathogenesis of Parkinson's disease (PD). The ability of alpha-syn oligomers to form ionic channels is postulated as a channelopathy mechanism in human brain. Here we identified a ganglioside-binding domain in alpha-syn (fragment 34-50), which includes the mutation site 46 linked to a familial form of PD (E46K). We show that this fragment is structurally related to the common glycosphingolipid-binding domain (GBD) shared by various microbial and amyloid proteins, including Alzheimer's beta-amyloid peptide. alpha-Syn GBD interacts with several glycosphingolipids but has a marked preference for GM3, a minor brain ganglioside whose expression increases with aging. The alpha-syn mutant E46K has a stronger affinity for GM3 than the wild-type protein, and the interaction is inhibited by 3'-sialyllactose (the glycone part of GM3). Alanine substitutions of Lys34 and Tyr39 in synthetic GBD peptides resulted in limited interaction with GM3, demonstrating the critical role of these residues in GM3 recognition. When incubated with reconstituted phosphatidylcholine bilayers, the E46K protein formed channels that are five times less conductive than those formed by wild-type alpha-syn, exhibit a higher selectivity for cations, and present an asymmetrical response to voltage and nonstop single-channel activity. This E46K-associated channelopathy was no longer observed when GM3 was present in phosphatidylcholine bilayers. This corrective effect was highly specific for GM3, since it was not obtained with the major brain ganglioside GM1 but was still detected in bilayer membranes containing both GM3 and GM1. Moreover, synthetic GBD peptides prevented the interaction of alpha-syn proteins with GM3, thus abolishing the regulatory effects of GM3 on alpha-syn-mediated channel formation. Overall, these data show that GM3 can specifically regulate alpha-syn-induced channel formation and raise the intriguing possibility that this minor brain ganglioside could play a key protective role in the pathogenesis of PD.

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De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins.

Publication Date: 2010 Mar 26 PMID: 20114051
Authors: Shi, L. - Wheeler, J. C. - Sweet, R. W. - Lu, J. - Luo, J. - Tornetta, M. - Whitaker, B. - Reddy, R. - Brittingham, R. - Borozdina, L. - Chen, Q. - Amegadzie, B. - Knight, D. M. - Almagro, J. C. - Tsui, P.
Journal: J Mol Biol

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.

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Repositioning of Transmembrane alpha-Helices during Membrane Protein Folding.

Publication Date: 2010 Mar 19 PMID: 20109468
Authors: Kauko, A. - Hedin, L. E. - Thebaud, E. - Cristobal, S. - Elofsson, A. - von Heijne, G.
Journal: J Mol Biol

We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt(Ph) glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.

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Labeling and Localization of the Herpes Simplex Virus Capsid Protein UL25 and Its Interaction with the Two Triplexes Closest to the Penton.

Publication Date: 2010 Mar 26 PMID: 20109467
Authors: Conway, J. F. - Cockrell, S. K. - Copeland, A. M. - Newcomb, W. W. - Brown, J. C. - Homa, F. L.
Journal: J Mol Biol

The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.

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Comparative Analysis of the Mosaic Genomes of Tailed Archaeal Viruses and Proviruses Suggests Common Themes for Virion Architecture and Assembly with Tailed Viruses of Bacteria.

Publication Date: 2010 Mar 19 PMID: 20109464
Authors: Krupovic, M. - Forterre, P. - Bamford, D. H.
Journal: J Mol Biol

Tailed double-stranded DNA viruses (order Caudovirales) represent the dominant morphotype among viruses infecting bacteria. Analysis and comparison of complete genome sequences of tailed bacterial viruses provided insights into their origin and evolution. Structural and genomic studies have unexpectedly revealed that tailed bacterial viruses are evolutionarily related to eukaryotic herpesviruses. Organisms from the third domain of life, Archaea, are also infected by viruses that, in their overall morphology, resemble tailed viruses of bacteria. However, high-resolution structural information is currently unavailable for any of these viruses, and only a few complete genomes have been sequenced so far. Here we identified nine proviruses that are clearly related to tailed bacterial viruses and integrated into chromosomes of species belonging to four different taxonomic orders of the Archaea. This more than doubled the number of genome sequences available for comparative studies. Our analyses indicate that highly mosaic tailed archaeal virus genomes evolve by homologous and illegitimate recombination with genomes of other viruses, by diversification, and by acquisition of cellular genes. Comparative genomics of these viruses and related proviruses revealed a set of conserved genes encoding putative proteins similar to virion assembly and maturation, as well as genome packaging proteins of tailed bacterial viruses and herpesviruses. Furthermore, fold prediction and structural modeling experiments suggest that the major capsid proteins of tailed archaeal viruses adopt the same topology as the corresponding proteins of tailed bacterial viruses and eukaryotic herpesviruses. Data presented in this study strongly support the hypothesis that tailed viruses infecting archaea share a common ancestry with tailed bacterial viruses and herpesviruses.

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Structure of the Membrane Domain of Human Erythrocyte Anion Exchanger 1 Revealed by Electron Crystallography.

Publication Date: 2010 Mar 19 PMID: 20100494
Authors: Yamaguchi, T. - Ikeda, Y. - Abe, Y. - Kuma, H. - Kang, D. - Hamasaki, N. - Hirai, T.
Journal: J Mol Biol

The membrane domain of human erythrocyte anion exchanger 1 (AE1) works as a Cl(-)/HCO(3)(-) antiporter. This exchange is a key step for CO(2)/O(2) circulation in the blood. In spite of their importance, structural information about AE1 and the AE (anion exchanger) family are still very limited. We used electron microscopy to solve the three-dimensional structure of the AE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-A resolution. A dimer of AE1 membrane domains packed in two-dimensional array showed a projection map similar to that of the prokaryotic homolog of the ClC chloride channel, a Cl(-)/H(+) antiporter. In a three-dimensional map, there are V-shaped densities near the center of the dimer and slightly narrower V-shaped clusters at a greater distance from the center of the dimer. These appear to be inserted into the membrane from opposite sides. The structural motifs, two homologous pairs of helices in internal repeats of the ClC transporter (helices B+C and J+K), are well fitted to those AE1 densities after simple domain movement.

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Surrobodies with Functional Tails.

Publication Date: 2010 Mar 19 PMID: 20100493
Authors: Xu, L. - Estelles, A. - Briante, R. - Kurtzman, A. L. - Hannum, C. H. - Kashyap, A. K. - Horowitz, L. - Horowitz, M. - Bhatt, R. R. - Lerner, R. A.
Journal: J Mol Biol

Surrobodies(2) are a unique type of binding protein based on the pre-B-cell receptor (pre-BCR). The pre-BCR is transiently expressed during development of the antibody repertoire. Unlike heterotetrameric canonical antibodies that are composed of identical pairs of heavy and light chains, the pre-BCR is a heterohexameric complex composed of identical pairs of heavy chains that are each paired with a two-subunit surrogate light chain (SLC). The SLC contains nonimmunoglobulin-like peptide extensions on each of the two SLC components. This arrangement provides unique opportunities for protein engineering by functional derivatization of these nonimmunoglobulin-like tails. Here we report recombinant fusions to these tails with either a fully active cytokine or with single-chain variable fragment (scFv) domains to generate Surrobodies with unique functions or Surrobodies that are bispecific with respect to targeted binding.

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Regulation of Oscillatory Contraction in Insect Flight Muscle by Troponin.

Publication Date: 2010 Mar 19 PMID: 20100491
Authors: Krzic, U. - Rybin, V. - Leonard, K. R. - Linke, W. A. - Bullard, B.
Journal: J Mol Biol

Insect indirect flight muscle is activated by sinusoidal length change, which enables the muscle to work at high frequencies, and contracts isometrically in response to Ca(2+). Indirect flight muscle has two TnC isoforms: F1 binding a single Ca(2+) in the C-domain, and F2 binding Ca(2+) in the N- and C-domains. Fibres substituted with F1 produce delayed force in response to a single rapid stretch, and those with F2 produce isometric force in response to Ca(2+). We have studied the effect of TnC isoforms on oscillatory work. In native Lethocerus indicus fibres, oscillatory work was superimposed on a level of isometric force that depended on Ca(2+) concentration. Maximum work was produced at pCa 6.1; at higher concentrations, work decreased as isometric force increased. In fibres substituted with F1 alone, work continued to rise as Ca(2+) was increased up to pCa 4.7. Fibres substituted with various F1:F2 ratios produced maximal work at a ratio of 100:1 or 50:1; a higher proportion of F2 increased isometric force at the expense of oscillatory work. The F1:F2 ratio was 9.8:1 in native fibres, as measured by immunofluorescence, using isoform-specific antibodies. The small amount of F2 needed to restore work to levels obtained for the native fibre is likely to be due to the relative affinity of F1 and F2 for TnH, the Lethocerus homologue of TnI. Affinity of TnC isoforms for a TnI fragment of TnH was measured by isothermal titration calorimetry. The K(d) was 1.01 muM for F1 binding and 22.7 nM for F2. The higher affinity of F2 can be attributed to two TnH binding sites on F2 and a single site on F1. Stretch may be sensed by an extended C-terminal domain of TnH, resulting in reversible dissociation of the inhibitory sequence from actin during the oscillatory cycle.

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Resonance Assignment and Three-Dimensional Structure Determination of a Human alpha-Defensin, HNP-1, by Solid-State NMR.

Publication Date: 2010 Mar 26 PMID: 20097206
Authors: Zhang, Y. - Doherty, T. - Li, J. - Lu, W. - Barinka, C. - Lubkowski, J. - Hong, M.
Journal: J Mol Biol

Human alpha-defensins [human neutrophil peptides (HNPs)] are immune defense mini-proteins that act by disrupting microbial cell membranes. Elucidating the three-dimensional (3D) structures of HNPs in lipid membranes is important for understanding their mechanisms of action. Using solid-state NMR (SSNMR), we have determined the 3D structure of HNP-1 in a microcrystalline state outside the lipid membrane, which provides benchmarks for structure determination and comparison with the membrane-bound state. From a suite of two-dimensional and 3D magic-angle spinning experiments, (13)C and (15)N chemical shifts that yielded torsion angle constraints were obtained, while inter-residue distances were obtained to restrain the 3D fold. Together, these constraints led to the first high-resolution SSNMR structure of a human defensin. The SSNMR structure has close similarity to the crystal structures of the HNP family, with the exception of the loop region between the first and second beta-strands. The difference, which is partially validated by direct torsion angle measurements of selected loop residues, suggests possible conformational variation and flexibility of this segment of the protein, which may regulate HNP interaction with the phospholipid membrane of microbial cells.

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Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus.

Publication Date: 2010 Mar 26 PMID: 20097205
Authors: Steiner, K. - Hagelueken, G. - Messner, P. - Schaffer, C. - Naismith, J. H.
Journal: J Mol Biol

Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by alpha-1,3-, alpha-1,2-, and beta-1,2-linkages. The formation of the beta-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-beta-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-beta-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition.

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Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy.

Publication Date: 2010 Mar 26 PMID: 20096706
Authors: Ikuta, K. - Yersin, A. - Ikai, A. - Aisen, P. - Kohgo, Y.
Journal: J Mol Biol

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, alpha and beta. Like TfR1, TfR2alpha is a type II membrane protein, but the beta form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2alpha, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2alpha is 5.6x10(6) M(-)(1), which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2alpha without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2alpha with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2alpha and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2alpha by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2alpha may help account for the different physiological roles of the two receptors.

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Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF.

Publication Date: 2010 Mar 19 PMID: 20096705
Authors: Schulz, E. C. - Schwarzer, D. - Frank, M. - Stummeyer, K. - Muhlenhoff, M. - Dickmanns, A. - Gerardy-Schahn, R. - Ficner, R.
Journal: J Mol Biol

An alpha-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports host infection by means of molecular mimicry. Bacteriophages of the K1 family, infecting E. coli K1, specifically recognize and degrade this polySia capsule utilizing tailspike endosialidases. While the crystal structure for the catalytic domain of the endosialidase of bacteriophage K1F (endoNF) has been solved, there is yet no structural information on the mode of polySia binding and cleavage available. The crystal structure of activity deficient active-site mutants of the homotrimeric endoNF cocrystallized with oligomeric sialic acid identified three independent polySia binding sites in each endoNF monomer. The bound oligomeric sialic acid displays distinct conformations at each site. In the active site, a Sia(3) molecule is bound in an extended conformation representing the enzyme-product complex. Structural and biochemical data supported by molecular modeling enable to propose a reaction mechanism for polySia cleavage by endoNF.

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Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin.

Publication Date: 2010 Mar 26 PMID: 20096704
Authors: Perez, A. C. - Karst, J. C. - Davi, M. - Guijarro, J. I. - Ladant, D. - Chenal, A.
Journal: J Mol Biol

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel beta-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a beta-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.

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Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein.

Publication Date: 2010 Mar 26 PMID: 20096703
Authors: Kozlov, G. - Menade, M. - Rosenauer, A. - Nguyen, L. - Gehring, K.
Journal: J Mol Biol

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.

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A Dynamic Model of HIV Integrase Inhibition and Drug Resistance.

Publication Date: 2010 Mar 26 PMID: 20096702
Authors: Perryman, A. L. - Forli, S. - Morris, G. M. - Burt, C. - Cheng, Y. - Palmer, M. J. - Whitby, K. - McCammon, J. A. - Phillips, C. - Olson, A. J.
Journal: J Mol Biol

Human immunodeficiency virus type 1 (HIV-1) integrase is one of three virally encoded enzymes essential for replication and, therefore, a rational choice as a drug target for the treatment of HIV-1-infected individuals. In 2007, raltegravir became the first integrase inhibitor approved for use in the treatment of HIV-infected patients, more than a decade since the approval of the first protease inhibitor (saquinavir, Hoffman La-Roche, 1995) and two decades since the approval of the first reverse transcriptase inhibitor (retrovir, GlaxoSmithKline, 1987). The slow progress toward a clinically effective HIV-1 integrase inhibitor can at least in part be attributed to a poor structural understanding of this key viral protein. Here we describe the development of a restrained molecular dynamics protocol that produces a more accurate model of the active site of this drug target. This model provides an advance on previously described models as it ensures that the catalytic DDE motif makes correct, monodentate interactions with the two active-site magnesium ions. Dynamic restraints applied to this coordination state create models with the correct solvation sphere for the metal ion complex and highlight the coordination sites available for metal-binding ligands. Application of appropriate dynamic flexibility to the core domain allowed the inclusion of multiple conformational states in subsequent docking studies. These models have allowed us to (1) explore the effects of key drug resistance mutations on the dynamic flexibility and conformational preferences of HIV integrase and to (2) study raltegravir binding in the context of these dynamic models of both wild type and the G140S/Q148H drug-resistant enzyme.

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Neutron Spin-Echo Studies of Hemoglobin and Myoglobin: Multiscale Internal Dynamics.

Publication Date: 2010 Mar 26 PMID: 20096701
Authors: Lal, J. - Fouquet, P. - Maccarini, M. - Makowski, L.
Journal: J Mol Biol

Neutron spin-echo spectroscopy was used to study structural fluctuations that occur in hemoglobin (Hb) and myoglobin (Mb) in solution. Using neutron spin-echo data up to a very high momentum transfer q ( approximately 0.62 A(-)(1)), we characterized the internal dynamics of these proteins at the levels of dynamic pair correlation function and self-correlation function in the time range of several picoseconds to a few nanoseconds. In the same protein solution, data transition from pair correlation motion to self-correlation motion as the momentum transfer q increases. At low q, coherent scattering dominates; at high q, observations are largely due to incoherent scattering. The low q data were interpreted in terms of an effective diffusion coefficient; on the other hand, the high q data were interpreted in terms of mean square displacements. Comparison of data from the two homologous proteins collected at different temperatures and protein concentrations was used to assess the contributions made by translational and rotational diffusion and internal modes of motion to the data. The temperature dependence of decay times can be attributed to changes in the viscosity and temperature of the solvent, as predicted by the Stokes-Einstein relationship. This is true for contributions from both diffusive and internal modes of motion, indicating an intimate relationship between the internal dynamics of the proteins and the viscosity of the solvent. Viscosity change associated with protein concentration can account for changes in diffusion observed at different concentrations, but is apparently not the only factor involved in the changes in internal dynamics observed with change in protein concentration. Data collected at high q indicate that internal modes in Mb are generally faster than those in Hb, perhaps due to the greater surface-to-volume ratio of Mb and the fact that surface groups tend to exhibit faster motion than buried groups. Comparison of data from Hb and data from Mb at low q indicates an unexpectedly rapid motion of Hb alphabeta dimers relative to one another. Dynamic motion of subunits is increasingly perceived as important to the allosteric behavior of Hb. Our data demonstrate that this motion is highly sensitive to protein concentration, temperature, and solvent viscosity, indicating that great care needs to be exercised in interpreting its effect on protein function.

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HK97 Maturation Studied by Crystallography and H/(2)H Exchange Reveals the Structural Basis for Exothermic Particle Transitions.

Publication Date: 2010 Mar 26 PMID: 20093122
Authors: Gertsman, I. - Komives, E. A. - Johnson, J. E.
Journal: J Mol Biol

HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of approximately 20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-A-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-A-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers.

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Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases.

Publication Date: 2010 Mar 19 PMID: 20083122
Authors: Tars, K. - Olin, B. - Mannervik, B.
Journal: J Mol Biol

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Delta(5)-androstene-3-17-dione into Delta(4)-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.

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RNA Polymerase II and TAFs Undergo a Slow Isomerization after the Polymerase Is Recruited to Promoter-Bound TFIID.

Publication Date: 2010 Mar 19 PMID: 20083121
Authors: Yakovchuk, P. - Gilman, B. - Goodrich, J. A. - Kugel, J. F.
Journal: J Mol Biol

Transcription of mRNA genes requires that RNA polymerase II (Pol II) and the general transcription factors assemble on promoter DNA to form an organized complex capable of initiating transcription. Biochemical studies have shown that Pol II and TFIID (transcription factor IID) contact overlapping regions of the promoter, leading to the question of how these large factors reconcile their promoter interactions during complex assembly. To investigate how the TAF (TATA-binding protein-associated factor) subunits of TFIID alter the kinetic mechanism by which complexes assemble on promoters, we used a highly purified human transcription system. We found that TAFs sharply decrease the rate at which Pol II, TFIIB, and TFIIF assemble on promoter-bound TFIID-TFIIA. Interestingly, the slow step in this process is not recruitment of these factors to the DNA, but rather a postrecruitment isomerization of protein-DNA contacts that occurs throughout the core promoter. Our findings support a model in which Pol II and the general transcription factors rapidly bind promoter-bound TFIID-TFIIA, after which complexes undergo a slow isomerization in which the TAFs reorganize their contacts with the promoter to allow Pol II to properly engage the DNA. In this manner, TAFs kinetically repress basal transcription.

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The C-terminal Lysine of Ogg2 DNA Glycosylases is a Major Molecular Determinant for Guanine/8-Oxoguanine Distinction.

Publication Date: 2010 Mar 19 PMID: 20083120
Authors: Faucher, F. - Wallace, S. S. - Doublie, S.
Journal: J Mol Biol

7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.

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Molecular dynamics reveal the essential role of linker motions in the function of cullin-RING E3 ligases.

Publication Date: 2010 Mar 12 PMID: 20083119
Authors: Liu, J. - Nussinov, R.
Journal: J Mol Biol

Tagging proteins by polyubiquitin is a key step in protein degradation. Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from the E2-conjugating enzyme to the substrate, yet crystallography indicates a large distance between the E2 and the substrate, raising the question of how this distance is bridged in the ubiquitin transfer reaction. Here, we demonstrate that the linker motions in the substrate binding proteins can allosterically shorten this distance to facilitate this crucial ubiquitin transfer step and increase this distance to allow polyubiquitination. We performed molecular dynamics simulations for five substrate binding proteins, Skp2, Fbw7, beta-TrCP1, Cdc4, and pVHL, in two forms: bound to their substrates and bound to both substrate and adaptor. The adaptor connects the substrate binding proteins to the cullin. In the bound-to-both forms of all cases, we observed rotations of the substrate binding domain, shortening the gap between the tip of the substrate peptide and the E2 active site by 7-12 A compared with the crystal structures. Overall, together with our earlier simulations of the unbound forms and the bound-to-adaptor forms, the emerging picture is that the maximum distance of 51-73 A between the substrate binding domain and the E2 active site in the modeled unbound forms of these five proteins shrinks to a minimum of 39-49 A in the bound-to-both forms. This large distance range, the result of allosterically controlled linker motions, facilitates ubiquitin transfer and polyubiquitination and as such argues that the cullin-RING E3 ubiquitin ligase is under conformational control. We further observed that substrate binding proteins with multiple substrate acceptor lysines have a larger distance range between the substrate and the E2 as compared with beta-TrCP1, with only one acceptor lysine.

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Molecular Analyses of an Unusual Translesion DNA Polymerase from Methanosarcina acetivorans C2A.

Publication Date: 2010 Mar 19 PMID: 20080107
Authors: Lin, L. J. - Yoshinaga, A. - Lin, Y. - Guzman, C. - Chen, Y. H. - Mei, S. - Lagunas, A. M. - Koike, S. - Iwai, S. - Spies, M. A. - Nair, S. K. - Mackie, R. I. - Ishino, Y. - Cann, I. K.
Journal: J Mol Biol

The domain Archaea is composed of several subdomains, and prominent among them are the Crenarchaeota and the Euryarchaeota. Biochemically characterized archaeal family Y DNA polymerases (Pols) or DinB homologs, to date, are all from crenarchaeal organisms, especially the genus Sulfolobus. Here, we demonstrate that archaeal family Y Pols fall into five clusters based on phylogenetic analysis. MacDinB-1, the homolog from the euryarchaeon Methanosarcina acetivorans that is characterized in this study, belongs to cluster II. Therefore, MacDinB-1 is different from the Sulfolobus DinB proteins, which are members of cluster I. In addition to translesion DNA synthesis activity, MacDinB-1 synthesized unusually long products ( approximately 7.2 kb) in the presence of its cognate proliferating cell nuclear antigen (PCNA). The PCNA-interacting site in MacDinB-1 was identified by mutational analysis in a C-terminally located heptapeptide akin to a PIP (PCNA-interacting protein) box. In vitro assays from the present report suggested that MacDinB-1 works in an error-free mode to repair cyclobutane pyrimidine dimers. This study on a euryarchaeal DinB homolog provides important insights into the functional diversity of the family Y Pols, and the availability of a genetic system for this archaeon should allow subsequent elucidation of the physiological significance of this enzyme in M. acetivorans cells.

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Different folding pathways taken by highly homologous proteins, goat alpha-lactalbumin and canine milk lysozyme.

Publication Date: 2010 Mar 12 PMID: 20080106
Authors: Nakamura, T. - Makabe, K. - Tomoyori, K. - Maki, K. - Mukaiyama, A. - Kuwajima, K.
Journal: J Mol Biol

Is the folding pathway conserved in homologous proteins? To address this question, we compared the folding pathways of goat alpha-lactalbumin and canine milk lysozyme using equilibrium and kinetic circular dichroism spectroscopy. Both Ca(2+)-binding proteins have 41% sequence identity and essentially identical backbone structures. The Phi-value analysis, based on the effect of Ca(2+) on the folding kinetics, showed that the Ca(2+)-binding site was well organized in the transition state in alpha-lactalbumin, although it was not yet organized in lysozyme. Equilibrium unfolding and hydrogen-exchange 2D NMR analysis of the molten globule intermediate also showed that different regions were stabilized in the two proteins. In alpha-lactalbumin, the Ca(2+)-binding site and the C-helix were weakly organized, whereas the A- and B-helices, both distant from the Ca(2+)-binding site, were well organized in lysozyme. The results thus provide an example of highly homologous proteins taking different folding pathways. To understand the molecular origin of this difference, we investigated the native three-dimensional structures of the proteins in terms of non-local contact clusters, a parameter based on the residue-residue contact map and known to be well correlated with the folding rate of non-two-state proteins. There were remarkable differences between the proteins in the distribution of the non-local contact clusters, and these differences provided a reasonable explanation of the observed difference in the folding initiation sites. In conclusion, the protein folding pathway is determined not only by the backbone topology but also by the specific side-chain interactions of contacting residues.

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Human Positive Coactivator 4 Controls Heterochromatinization and Silencing of Neural Gene Expression by Interacting with REST/NRSF and CoREST.

Publication Date: 2010 Mar 19 PMID: 20080105
Authors: Das, C. - Gadad, S. S. - Kundu, T. K.
Journal: J Mol Biol

The highly abundant, multifunctional transcriptional positive coactivator 4 (PC4) plays important roles in transcription, replication and DNA repair. Our recent work showed that PC4 is a bona fide non-histone component of chromatin. Here, we report that knockdown of PC4 dramatically alters heterochromatin organization of the genome, accompanied by increased H3K9 (histone H3 at lysine residue 9)/14 acetylation, H3K4 trimethylation and reduction in the level of H3K9 dimethylation. These posttranslational modifications of histone H3 result in overexpression of normally silenced genes (e.g., neural genes) located in heterochromatin. The results of ChIP (chromatin immunoprecipitation) and re-ChIP assays showed that overexpression of a neuronal-specific gene is accompanied by histone hyperacetylation. We further show that PC4 interacts with heterochromatin protein 1alpha, REST/NRSF (RE1-silencing transcription factor/neuron-restrictive silencer factor) and CoREST to establish the repressed state of neural genes in nonneuronal cells. Thus, PC4 plays a crucial role in maintaining a dynamic chromatin state and heterochromatin gene silencing.

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The Single-Stranded DNA Binding Protein of Sulfolobus solfataricus Acts in the Presynaptic Step of Homologous Recombination.

Publication Date: 2010 Mar 19 PMID: 20080104
Authors: Rolfsmeier, M. L. - Haseltine, C. A.
Journal: J Mol Biol

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.

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Interactions between PTB RRMs Induce Slow Motions and Increase RNA Binding Affinity.

Publication Date: 2010 Mar 19 PMID: 20080103
Authors: Maynard, C. M. - Hall, K. B.
Journal: J Mol Biol

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. (15)N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.

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Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and Final Hairpin Conformations.

Publication Date: 2010 Mar 19 PMID: 20080102
Authors: Sackett, K. - Nethercott, M. J. - Epand, R. F. - Epand, R. M. - Kindra, D. R. - Shai, Y. - Weliky, D. P.
Journal: J Mol Biol

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The approximately 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large "N70" (gp41 1-70) and "FP-Hairpin" constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter "FP34" (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either beta-sheet or helical structure with greater beta-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.

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Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond.

Publication Date: 2010 Mar 19 PMID: 20080101
Authors: Podzelinska, K. - Latimer, R. - Bhattacharya, A. - Vining, L. C. - Zechel, D. L. - Jia, Z.
Journal: J Mol Biol

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 A resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4alpha)-OOH intermediate. Strikingly, the 8alpha carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

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Crystal Structure of Mouse Elf3 C-terminal DNA-binding Domain in Complex with Type II TGF-beta Receptor Promoter DNA.

Publication Date: 2010 Mar 19 PMID: 20079749
Authors: Agarkar, V. B. - Babayeva, N. D. - Wilder, P. J. - Rizzino, A. - Tahirov, T. H.
Journal: J Mol Biol

The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-beta receptor gene (TbetaR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 A resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-beta receptor promoter DNA (mTbetaR-II(DNA)). Elf3 contacts the core GGAA motif of the B-site from a major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.

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Crystal Structure of Escherichia coli Enterobactin-specific Isochorismate Synthase (EntC) Bound to its Reaction Product Isochorismate: Implications for the Enzyme Mechanism and Differential Activity of Chorismate-utilizing Enzymes.

Publication Date: 2010 Mar 19 PMID: 20079748
Authors: Sridharan, S. - Howard, N. - Kerbarh, O. - Blaszczyk, M. - Abell, C. - Blundell, T. L.
Journal: J Mol Biol

EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg(2+)at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex alpha+beta fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.

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Four distinct structural domains in Clostridium difficile toxin B visualized using SAXS.

Publication Date: 2010 Mar 12 PMID: 20070948
Authors: Albesa-Jove, D. - Bertrand, T. - Carpenter, E. P. - Swain, G. V. - Lim, J. - Zhang, J. - Haire, L. F. - Vasisht, N. - Braun, V. - Lange, A. - von Eichel-Streiber, C. - Svergun, D. I. - Fairweather, N. F. - Brown, K. A.
Journal: J Mol Biol

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.

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Estrogen increases expression of the human prostacyclin receptor within the vasculature through an ERalpha-dependent mechanism.

Publication Date: 2010 Feb 26 PMID: 20070947
Authors: Turner, E. C. - Kinsella, B. T.
Journal: J Mol Biol

Prostacyclin and the prostacyclin receptor (IP) are implicated in mediating many of the atheroprotective effects of estrogen in both humans and in animal models but through unknown mechanisms. Hence, herein the influence of estrogen on IP gene expression in endothelial EA.hy926, human erythroleukemia 92.1.7 and primary human aortic smooth muscle cells was investigated. Estrogen increased hIP mRNA levels, promoter (PrmIP)-directed reporter gene expression and cicaprost-dependent cAMP generation in all cell types, effects that were abrogated by actinomycin D and the general estrogen receptor (ER)-alpha/ERbeta antagonist ICI 182,780. Furthermore, the ERalpha-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), but not the ERbeta-agonist 2,3-bis(4-hydroxyphenyl)-propionitrile, significantly increased hIP mRNA and PrmIP-directed gene expression. Deletional and mutational analysis of PrmIP uncovered an evolutionary conserved estrogen-response element, while electrophoretic mobility shift, antibody-supershift and chromatin immunoprecipitations assays confirmed the direct binding of ERalpha, but not ERbeta, to PrmIP both in vitro and in vivo. Moreover, immunofluorescence microscopy corroborated that estrogen and PPT increased hIP expression in primary human aortic smooth muscle cells. In conclusion, the hIP gene is directly regulated by estrogen that largely occurs through an ERalpha-dependent transcriptional mechanism and thereby provides critical insights into the role of prostacyclin/hIP in mediating the atheroprotective effects of estrogen within the human vasculature.

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The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.

Publication Date: 2010 Mar 5 PMID: 20070946
Authors: Castrec, B. - Laurent, S. - Henneke, G. - Flament, D. - Raffin, J. P.
Journal: J Mol Biol

A glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D.

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TRPM7 activates m-calpain by stress-dependent stimulation of p38 MAPK and c-Jun N-terminal kinase.

Publication Date: 2010 Mar 5 PMID: 20070945
Authors: Su, L. T. - Chen, H. C. - Gonzalez-Pagan, O. - Overton, J. D. - Xie, J. - Yue, L. - Runnels, L. W.
Journal: J Mol Biol

TRPM7 is a Ca(2)(+)-permeant and Mg(2)(+)-permeant ion channel in possession of its own kinase domain. In a previous study, we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion, which was dependent on the Ca(2+)-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca(2+). Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide, causing the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg(2+); however, when the concentration of either external Ca(2+) or Mg(2+) was increased to favor the permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stresses, producing cell rounding mediated by p38 MAPK/JNK-dependent activation of m-calpain.

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The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase.

Publication Date: 2010 Mar 12 PMID: 20070944
Authors: Larson, E. T. - Mudeppa, D. G. - Gillespie, J. R. - Mueller, N. - Napuli, A. J. - Arif, J. A. - Ross, J. - Arakaki, T. L. - Lauricella, A. - Detitta, G. - Luft, J. - Zucker, F. - Verlinde, C. L. - Fan, E. - Van Voorhis, W. C. - Buckner, F. S. - Rathod, P. K. - Hol, W. G. - Merritt, E. A.
Journal: J Mol Biol

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.

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Insights into higher-order organization of the cellulosome revealed by a dissect-and-build approach: crystal structure of interacting Clostridium thermocellum multimodular components.

Publication Date: 2010 Mar 5 PMID: 20070943
Authors: Adams, J. J. - Currie, M. A. - Ali, S. - Bayer, E. A. - Jia, Z. - Smith, S. P.
Journal: J Mol Biol

Cellulosomes are large, multienzyme, plant cell wall-degrading protein complexes found affixed to the surface of a variety of anaerobic microbes. The core of the cellulosome is a noncatalytic scaffoldin protein, which contains several type-I cohesin modules that bind type-I dockerin-containing enzymatic subunits, a cellulose-binding module, an X module, and a type-II dockerin that interacts with type-II cohesin-containing cell surface proteins. The unique arrangement of the enzymatic subunits in the cellulosome complex, made possible by the scaffoldin subunit, promotes enhanced substrate degradation relative to the enzymes free in solution. Despite representative high-resolution structures of all of the individual modules of the cellulosome, this mechanism of enzymatic synergy remains poorly understood. Consequently, a model of the entire cellulosome and a detailed picture of intermodular contacts will provide more detailed insight into cellulosome activity. Toward this goal, we have solved the structure of a multimodular heterodimeric complex from Clostridium thermocellum composed of the type-II cohesin module of the cell surface protein SdbA bound to a trimodular C-terminal fragment of the scaffoldin subunit CipA to a resolution of 1.95 A. The linker that connects the ninth type-I cohesin module and the X module has elevated temperature factors, reflecting an inherent flexibility within this region. Interestingly, a novel dimer interface was observed between CipA and a second, symmetry-related CipA molecule within the crystal structure, mediated by contacts between a type-I cohesin and an X module of a symmetry mate, resulting in two intertwined scaffoldins. Sedimentation velocity experiments confirmed that dimerization also occurs in solution. These observations support the intriguing possibility that individual cellulosomes can associate with one another via inter-scaffoldin interactions, which may play a role in the mechanism of action of the complex.

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DNA replication-coupled PCNA mono-ubiquitination and polymerase switching in a human in vitro system.

Publication Date: 2010 Feb 26 PMID: 20064529
Authors: Masuda, Y. - Piao, J. - Kamiya, K.
Journal: J Mol Biol

Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol delta on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol delta is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol delta and a translesion pol, pol eta, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol eta. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.

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RAB6C Is a Retrogene that Encodes a Centrosomal Protein Involved in Cell Cycle Progression.

Publication Date: 2010 Mar 19 PMID: 20064528
Authors: Young, J. - Menetrey, J. - Goud, B.
Journal: J Mol Biol

Rab-GTPases are key regulators of membrane transport, and growing evidence indicates that their expression levels are altered in certain human malignancies, including cancer. Rab6C, a newly identified Rab6 subfamily member, has attracted recent attention because its reduced expression might confer a selective advantage to drug-resistant breast cancer cells. Here, we report that RAB6C is a primate-specific retrogene derived from a RAB6A' transcript. RAB6C is transcribed in a limited number of human tissues including brain, testis, prostate, and breast. Endogenous Rab6C is considerably less abundant and has a much shorter half-life than Rab6A'. Comparison of the GTP-binding motifs of Rab6C and Rab6A', homology modeling, and GTP-blot overlay assays indicate that amino acid changes in Rab6C have greatly reduced its GTP-binding affinity. Instead, the noncanonical GTP-binding domain of Rab6C mediates localization of the protein to the centrosome. Overexpression of Rab6C results in G1 arrest, and its specific depletion generates tetraploid cells with supernumerary centrosomes, revealing a role of Rab6C in events related to the centrosome and cell cycle progression. Thus, RAB6C is a rare example of a recently emerged retrogene that has acquired the status of a new gene, encoding a functional protein with altered characteristics compared to Rab6A'.

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Crystal structure of the ATP-dependent maturation factor of Ni,Fe-containing carbon monoxide dehydrogenases.

Publication Date: 2010 Mar 5 PMID: 20064527
Authors: Jeoung, J. H. - Giese, T. - Grunwald, M. - Dobbek, H.
Journal: J Mol Biol

CooC proteins are ATPases involved in the incorporation of nickel into the complex active site ([Ni-4Fe-4S]) cluster of Ni,Fe-dependent carbon monoxide dehydrogenases. The genome of the carboxydotrophic bacterium Carboxydothermus hydrogenoformans encodes five carbon monoxide dehydrogenases and three CooC-type proteins, of which CooC1 was shown to be a nickel-binding ATPase. We determined the crystal structure of CooC1 in four different states: empty, ADP-bound, Zn(2+)/ADP-bound, and Zn(2+)-bound. The structure of CooC1 consists of two spatially separated functional modules: an ATPase module containing the deviant Walker A motif and a metal-binding module that confers the specific function of CooC1. The ATPase module is homologous to other members of the MinD family and, in analogy to the dimeric structure of ATP-bound Soj, is likely responsible for the ATP-dependent dimerization of CooC1. Its core topology classifies CooC1 as a member of the MinD family of SIMIBI (signal recognition particle, MinD and BioD)-class NTPases. The crystal structure of Zn(2+)-bound CooC1 reveals a conserved C-X-C motif as the metal-binding site responsible for metal-induced dimerization. The competitive binding of Ni(2+) and Zn(2+) to CooC1 in solution confirms that the conserved C-X-C motif is also responsible for the interaction with Ni(2+). A comparison of the different CooC1 structures determined suggests a mutual dependence of metal-binding site and nucleotide-binding site.

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The RING domain of TRAF2 plays an essential role in the inhibition of TNFalpha-induced cell death but not in the activation of NF-kappaB.

Publication Date: 2010 Feb 26 PMID: 20064526
Authors: Zhang, L. - Blackwell, K. - Shi, Z. - Habelhah, H.
Journal: J Mol Biol

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of kappaB kinase (IKK), as well as in inhibiting apoptosis induced by TNFalpha. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFalpha-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-DeltaR) has been widely used as a dominant negative in transient overexpression systems to block TNFalpha-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-DeltaR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFalpha-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells efficiently inhibited the TNFalpha-induced later phase of prolonged JNK activation, yet failed to inhibit TNFalpha-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-DeltaR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFalpha-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFalpha and is essential for suppressing the noncanonical nuclear factor kappaB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFalpha-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFalpha-induced cell death.

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Comparative Genomic Analysis of 60 Mycobacteriophage Genomes: Genome Clustering, Gene Acquisition, and Gene Size.

Publication Date: 2010 Mar 19 PMID: 20064525
Authors: Hatfull, G. F. - Jacobs-Sera, D. - Lawrence, J. G. - Pope, W. H. - Russell, D. A. - Ko, C. C. - Weber, R. J. - Patel, M. C. - Germane, K. L. - Edgar, R. H. - Hoyte, N. N. - Bowman, C. A. - Tantoco, A. T. - Paladin, E. C. - Myers, M. S. - Smith, A. L. - Grace, M. S. - Pham, T. T. - O'Brien, M. B. - Vogelsberger, A. M. - Hryckowian, A. J. - Wynalek, J. L. - Donis-Keller, H. - Bogel, M. W. - Peebles, C. L. - Cresawn, S. G. - Hendrix, R. W.
Journal: J Mol Biol

Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60-all infecting a common bacterial host-provides further insight into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.

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Quorum sensing regulation in Aeromonas hydrophila.

Publication Date: 2010 Mar 5 PMID: 20064524
Authors: Garde, C. - Bjarnsholt, T. - Givskov, M. - Jakobsen, T. H. - Hentzer, M. - Claussen, A. - Sneppen, K. - Ferkinghoff-Borg, J. - Sams, T.
Journal: J Mol Biol

We present detailed results on the C4-HSL-mediated quorum sensing (QS) regulatory system of the opportunistic Gram-negative bacterium Aeromonas hydrophila. This bacterium contains a particularly simple QS system that allows for a detailed modeling of kinetics. In a model system (i.e., the Escherichia coli monitor strain MH205), the C4-HSL production of A. hydrophila is interrupted by fusion of gfp(ASV). In the present in vitro study, we measure the response of the QS regulatory ahyRI locus in the monitor strain to predetermined concentrations of C4-HSL signal molecules. A minimal kinetic model describes the data well. It can be solved analytically, providing substantial insight into the QS mechanism: at high concentrations of signal molecules, a slow decay of the activated regulator sets the timescale for the QS regulation loop. Slow saturation ensures that, in an A. hydrophila cell, the QS system is activated only by signal molecules produced by other A. hydrophila cells. Separate information on the ahyR and ahyI loci can be extracted, thus allowing the probe to be used in identifying the target when testing QS inhibitors.

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Functional Interaction of the Ras Effector RASSF5 with the Tyrosine Kinase Lck: Critical Role in Nucleocytoplasmic Transport and Cell Cycle Regulation.

Publication Date: 2010 Mar 19 PMID: 20064523
Authors: Kumari, G. - Singhal, P. K. - Suryaraja, R. - Mahalingam, S.
Journal: J Mol Biol

RASSF5 is a member of the Ras association domain family, which is known to be involved in cell growth regulation. Expression of RASSF5 is extinguished selectively by epigenetic mechanism(s) in different cancers and cell lines, and reexpression usually suppresses cell proliferation and tumorigenicity. To date, the mechanism regulating RASSF5 nuclear transport and its role in cell growth regulation remains unclear. Using heterokaryon assay, we have demonstrated that RASSF5 shuttles between the nucleus and the cytoplasm, and its export from the nucleus is sensitive to leptomycin B, suggesting that RASSF5 is exported from the nucleus by a CRM-1-dependent export pathway. We further demonstrate that RASSF5 contains a hydrophobic-rich nuclear export signal (NES) towards the C-terminus and two nuclear localization signals-one each at the N-terminus and the C-terminus. Combination of mutational and immunofluorescence analyses suggests that the functional NES residing between amino acids 260 and 300 in the C-terminus is necessary for the efficient export of RASSF5 from the nucleus. In addition, substitution of conserved hydrophobic residues within the minimal NES impaired RASSF5 export from the nucleus. Furthermore, exchange of proline residues within the putative Src homology 3 binding motifs altered the export of RASSF5 from the nucleus despite the presence of functional NES, suggesting that multiple domains independently modulate the nucleocytoplasmic transport of RASSF5. Interestingly, the present investigation provided evidence that RASSF5 interacts with the tyrosine kinase Lck through its C-terminal Src homology 2 binding motif and showed that Lck-mediated phosphorylation is critical for the efficient translocation of RASSF5 into the nuclear compartment. Interestingly, our data demonstrate that wild type and nuclear export defective (DeltaNES) mutant of RASSF5 but not the import defective mutant of accumulate the cells at G1/S phase and induce apoptosis. Furthermore, the Lck-interaction-defective mutant of RASSF5 induces apoptosis without altering cell cycle progression, suggesting that RASSF5 induces apoptosis independent of cell cycle arrest. Together, our data demonstrate that interaction with Lck is critical for RASSF5 phosphorylation, which in turn regulates the cell growth control activity of RASSF5. Finally, we have shown that RASSF5 encodes four splice variants and is translocated to the nucleus by the classical nuclear import pathway. One of the splice variants, RASSF5C, was found to be localized in the cytoplasm and translocated into the nucleus upon leptomycin B treatment despite the absence of N-terminal nuclear localization signal, suggesting that distribution of RASSF5 variants in different cellular compartments may be critical for Ras-dependent cell growth regulation. Collectively, the present investigation provided evidence that Lck-mediated phosphorylation regulates the nucleocytoplasmic shuttling and cell growth control activities of RASSF5.

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The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection.

Publication Date: 2010 Mar 5 PMID: 20060839
Authors: Elantak, L. - Wagner, S. - Herrmannova, A. - Karaskova, M. - Rutkai, E. - Lukavsky, P. J. - Valasek, L.
Journal: J Mol Biol

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix alpha1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding "pocket" residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

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A folding zone in the ribosomal exit tunnel for Kv1.3 helix formation.

Publication Date: 2010 Mar 12 PMID: 20060838
Authors: Tu, L. W. - Deutsch, C.
Journal: J Mol Biol

Although it is now clear that protein secondary structure can be acquired early, while the nascent peptide resides within the ribosomal exit tunnel, the principles governing folding of native polytopic proteins have not yet been elucidated. We now report an extensive investigation of native Kv1.3, a voltage-gated K(+) channel, including transmembrane and linker segments synthesized in sequence. These native segments form helices vectorially (N- to C-terminus) only in a permissive vestibule located in the last 20 A of the tunnel. Native linker sequences similarly fold in this vestibule. Finally, secondary structure acquired in the ribosome is retained in the translocon. These findings emerge from accessibility studies of a diversity of native transmembrane and linker sequences and may therefore be applicable to protein biogenesis in general.

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Crystal structure of a full-length autotransporter.

Publication Date: 2010 Feb 26 PMID: 20060837
Authors: van den Berg, B.
Journal: J Mol Biol

The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the beta-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the beta-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 A. EstA has a relatively narrow, 12-stranded beta-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the beta-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by alpha-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

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An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: structural, biochemical, and gene expression analysis.

Publication Date: 2010 Mar 12 PMID: 20060836
Authors: Chim, N. - Riley, R. - The, J. - Im, S. - Segelke, B. - Lekin, T. - Yu, M. - Hung, L. W. - Terwilliger, T. - Whitelegge, J. P. - Goulding, C. W.
Journal: J Mol Biol

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here, we present structural, biochemical, and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pK(a) for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein coexpression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicates anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions.

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The structure of the Salmonella typhimurium type III secretion system needle shows divergence from the flagellar system.

Publication Date: 2010 Mar 12 PMID: 20060835
Authors: Galkin, V. E. - Schmied, W. H. - Schraidt, O. - Marlovits, T. C. - Egelman, E. H.
Journal: J Mol Biol

The type III secretion system (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of approximately 5.6 subunits/turn, which is quite similar to that of the most intensively studied flagellar filament (from Salmonella typhimurium), which has approximately 5.5 subunits/turn. We now show that the Sa. typhimurium needle, expected by homology arguments to be more similar to the Sa. typhimurium flagellar filament than is the needle from Shigella, actually has approximately 6.3 subunits/turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the Sa. typhimurium flagellar filament, which shows a nearly crystalline order, the Sa. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact.

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Desolvation and development of specific hydrophobic core packing during Im7 folding.

Publication Date: 2010 Mar 12 PMID: 20053361
Authors: Bartlett, A. I. - Radford, S. E.
Journal: J Mol Biol

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val-->Thr or Phe-->Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Phi-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (>2.5 kJ mol(-1)) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.

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The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography.

Publication Date: 2010 Mar 12 PMID: 20053360
Authors: Charpentier, T. H. - Thompson, L. E. - Liriano, M. A. - Varney, K. M. - Wilder, P. T. - Pozharski, E. - Toth, E. A. - Weber, D. J.
Journal: J Mol Biol

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca(2)(+)-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca(2+)-S100B (1.5-A resolution) and S100B-Ca(2)(+)-TRTK-12 (2.0-A resolution) determined here indicate that the S100B-Ca(2+)-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca(2+)-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca(2)(+)-p53 peptide complex, TRTK-12 binding to Ca(2+)-S100B was found to increase the protein's Ca(2)(+)-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca(2+) ligands and/or improved the Ca(2+) coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca(2+)-TRTK-12 and S100B-Ca(2+) were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca(2+)-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR (15)N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca(2+)-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca(2+)-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.

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Conformational dynamics and structural plasticity play critical roles in the ubiquitin recognition of a UIM domain.

Publication Date: 2010 Mar 5 PMID: 20053359
Authors: Sgourakis, N. G. - Patel, M. M. - Garcia, A. E. - Makhatadze, G. I. - McCallum, S. A.
Journal: J Mol Biol

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single alpha-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of (15)N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.

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Structure, dynamics, and RNA interaction analysis of the human SBDS protein.

Publication Date: 2010 Mar 5 PMID: 20053358
Authors: de Oliveira, J. F. - Sforca, M. L. - Blumenschein, T. M. - Goldfeder, M. B. - Guimaraes, B. G. - Oliveira, C. C. - Zanchin, N. I. - Zeri, A. C.
Journal: J Mol Biol

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS.

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The dimeric structure and the bivalent recognition of H3K4me3 by the tumor suppressor ING4 suggests a mechanism for enhanced targeting of the HBO1 complex to chromatin.

Publication Date: 2010 Mar 5 PMID: 20053357
Authors: Palacios, A. - Moreno, A. - Oliveira, B. L. - Rivera, T. - Prieto, J. - Garcia, P. - Fernandez-Fernandez, M. R. - Bernado, P. - Palmero, I. - Blanco, F. J.
Journal: J Mol Biol

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at position K4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus in the five members of the ING family. ING4 facilitates histone H3 acetylation by the HBO1 complex. Here, we show that ING4 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING4, which contains the nuclear localization sequence, is disordered and flexible and does not directly interact with p53, or does it with very low affinity, in contrast to previous findings. The NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule. The detailed NMR analysis of the full-length dimeric protein binding to histone H3K4me3 shows essentially the same binding site and affinity as the isolated PHD finger. Therefore, the ING4 dimer has two identical and independent binding sites for H3K4me3 tails, which, in the context of the chromatin, could belong to the same or to different nucleosomes. These results show that ING4 is a bivalent reader of the chromatin H3K4me3 modification and suggest a mechanism for enhanced targeting of the HBO1 complex to specific chromatin sites. This mechanism could be common to other ING-containing remodeling complexes.

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Sequence recognition of DNA by protein-induced conformational transitions.

Publication Date: 2010 Mar 5 PMID: 20053356
Authors: Watkins, D. - Mohan, S. - Koudelka, G. B. - Williams, L. D.
Journal: J Mol Biol

The binding of proteins to specific sequences of DNA is an important feature of virtually all DNA transactions. Proteins recognize specific DNA sequences using both direct readout (sensing types and positions of DNA functional groups) and indirect readout (sensing DNA conformation and deformability). Previously we showed that the P22 c2 repressor N-terminal domain (P22R NTD) forces the central non-contacted 5'-ATAT-3' sequence of the DNA operator into the B' state, a state known to affect DNA hydration, rigidity and bending. Usually the B' state, with a narrow minor groove and a spine of hydration, is reserved for A-tract DNA (TpA steps disrupt A-tracts). Here, we have co-crystallized P22R NTD with an operator containing a central 5'-ACGT-3' sequence in the non-contacted region. C.G base pairs have not previously been observed in the B' state and are thought to prevent it. However, P22R NTD induces a narrow minor groove and a spine of hydration to 5'-ACGT-3'. We observe that C.G base pairs have distinctive destabilizing and disordering effects on the spine of hydration. It appears that the reduced stability of the spine results in a higher energy cost for the B to B' transition. The differential effect of DNA sequence on the barrier to this transition allows the protein to sense the non-contacted DNA sequence.

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Rapid structural characterization of human antibody-antigen complexes through experimentally validated computational docking.

Publication Date: 2010 Mar 12 PMID: 20053355
Authors: Simonelli, L. - Beltramello, M. - Yudina, Z. - Macagno, A. - Calzolai, L. - Varani, L.
Journal: J Mol Biol

If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition. To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution. As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus.

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Glycine-rich loop of mitochondrial processing peptidase alpha-subunit is responsible for substrate recognition by a mechanism analogous to mitochondrial receptor Tom20.

Publication Date: 2010 Mar 12 PMID: 20053354
Authors: Dvorakova-Hola, K. - Matuskova, A. - Kubala, M. - Otyepka, M. - Kucera, T. - Vecer, J. - Herman, P. - Parkhomenko, N. - Kutejova, E. - Janata, J.
Journal: J Mol Biol

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) alpha-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast alpha-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility. Moreover, time-resolved measurements showed that a functionless alpha-MPP with a single-residue deletion in the loop (Y303W/DeltaG292) is defective particularly in the primary contact with substrate. Thus, the GRL was proved to be part of a contact site of the enzyme specifically recognizing the substrate. Regarding the surface exposure and presence of the hydrophobic patches within the GRL, we proposed a functional analogy between the presequence recognition by the hydrophobic binding groove of the Tom20 mitochondrial import receptor and the GRL of the alpha-MPP. A molecular dynamics (MD) simulation of the MPP-substrate peptide complex model was employed to test this hypothesis. The initial positioning and conformation of the substrate peptide in the model fitting were chosen based on the analogy of its interaction with the Tom20 binding groove. MD simulation confirmed the stability of the proposed interaction and showed also a decrease in GRL flexibility in the presence of substrate, in agreement with fluorescence measurements. Moreover, conserved substrate hydrophobic residues in positions +1 and -4 to the cleavage site remain in close contact with the side chains of the GRL during the entire production part of MD simulation as stabilizing points of the hydrophobic interaction. We conclude that the GRL of the MPP alpha-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.

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Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension.

Publication Date: 2010 Mar 12 PMID: 20053353
Authors: Schushan, M. - Xiang, M. - Bogomiakov, P. - Padan, E. - Rao, R. - Ben-Tal, N.
Journal: J Mol Biol

Human NHA2 is a poorly characterized Na(+)/H(+) antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.

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Assembly of a 20-nm protein cage by Escherichia coli 2-hydroxypentadienoic acid hydratase.

Publication Date: 2010 Mar 12 PMID: 20053352
Authors: Montgomery, M. G. - Coker, A. R. - Taylor, I. A. - Wood, S. P.
Journal: J Mol Biol

The pentameric Escherichia coli enzyme 2-hydroxypentadienoic acid hydratase assembles to form a 20-nm-diameter particle comprising 60 protein subunits, arranged with 532 symmetry when crystallised at low pH in the presence of phosphate or sulphate ions. The particles form rapidly and are stable in solution during gel filtration at low pH. They are probably formed through trimers of pentamers, which are stabilised by the interaction of two phosphate ions with residues of the N-terminal domains of subunits at the 3-fold axis. Once the particles are formed at high concentrations of phosphate (or sulphate), they remain stable in solution at 20-fold lower concentrations of the anion. Guest molecules can be trapped within the hollow protein shell during assembly. The C-termini of the subunits are freely accessible on the surface of the protein cage and thus are ideal sites for addition of affinity tags or other modifications. These particles offer a convenient model system for studying the assembly of large symmetrical structures and a novel protein nanoparticle for encapsulation and cargo delivery.

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Engineering fully human monoclonal antibodies from murine variable regions.

Publication Date: 2010 Mar 12 PMID: 20045416
Authors: Bernett, M. J. - Karki, S. - Moore, G. L. - Leung, I. W. - Chen, H. - Pong, E. - Nguyen, D. H. - Jacinto, J. - Zalevsky, J. - Muchhal, U. S. - Desjarlais, J. R. - Lazar, G. A.
Journal: J Mol Biol

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.

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Interrupted catalysis: the EF4 (LepA) effect on back-translocation.

Publication Date: 2010 Mar 5 PMID: 20045415
Authors: Liu, H. - Pan, D. - Pech, M. - Cooperman, B. S.
Journal: J Mol Biol

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis.

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Slowing bacterial translation speed enhances eukaryotic protein folding efficiency.

Publication Date: 2010 Mar 12 PMID: 20043920
Authors: Siller, E. - DeZwaan, D. C. - Anderson, J. F. - Freeman, B. C. - Barral, J. M.
Journal: J Mol Biol

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.

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The humanness of macaque antibody sequences.

Publication Date: 2010 Mar 12 PMID: 20043919
Authors: Thullier, P. - Huish, O. - Pelat, T. - Martin, A. C.
Journal: J Mol Biol

Chimeric, humanized and human antibodies have successively been exploited as therapeutics because their increasing human ('self') character is expected to correspond with decreased immunogenicity, which is critical for their clinical development. Thus, humanness has been inferred to predict antibody immunogenicity. Humanness of antibody variable regions (V-regions) has recently been studied using a parameter (here referred to as the H-score) that evaluates similarity to expressed human sequences. Macaque (Macaca fascicularis) antibody sequences are of particular interest because they have been suggested to have extremely human-like character and, recently, macaque single-chain variable fragments with very high affinity for various antigens have been isolated. In this study, the H-scores of all macaque antibody V-regions available in sequence data banks were compared with those of their human counterparts using statistical tests. The results were found to be influenced by the relative size of the human families to which the macaque V-regions are related. As the relevance of families to immunogenicity is suspected but unproven, a new parameter (the 'G-score') was derived from the H-score to avoid this influence, and macaque V-region sequences were reanalyzed using the G-score. Both parameters show that these regions cannot be regarded as human when they derive from heavy chains, but the humanness of light chains is variable. It was shown that 'germline humanization' of a macaque V-region favourably influenced its humanness, as evaluated by both H-score and G-score. In addition, the humanness of macaque sequences presented in patents has been analyzed. The H-score and G-score define objectively the humanness of antibody V-regions, and their use is exemplified here.

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Substitution of Glu122 by glutamine revealed the function of the second water molecule as a proton donor in the binuclear metal enzyme creatininase.

Publication Date: 2010 Mar 5 PMID: 20043918
Authors: Yamashita, K. - Nakajima, Y. - Matsushita, H. - Nishiya, Y. - Yamazawa, R. - Wu, Y. F. - Matsubara, F. - Oyama, H. - Ito, K. - Yoshimoto, T.
Journal: J Mol Biol

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity. We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl(2) or MnCl(2). In the E122Q mutant, k(cat) is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.

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Energetic frustration of apomyoglobin folding: role of the B helix.

Publication Date: 2010 Mar 12 PMID: 20043917
Authors: Nishimura, C. - Dyson, H. J. - Wright, P. E.
Journal: J Mol Biol

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.

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Helix dynamics in LacY: helices II and IV.

Publication Date: 2010 Feb 26 PMID: 20043916
Authors: Liu, Z. - Madej, M. G. - Kaback, H. R.
Journal: J Mol Biol

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H+ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within approximately 4.2 A of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.

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Neutron diffraction studies of a class A beta-lactamase Toho-1 E166A/R274N/R276N triple mutant.

Publication Date: 2010 Mar 5 PMID: 20036259
Authors: Tomanicek, S. J. - Blakeley, M. P. - Cooper, J. - Chen, Y. - Afonine, P. V. - Coates, L.
Journal: J Mol Biol

beta-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the beta-lactam antibiotics is the production of beta-lactamase enzymes that inactivate beta-lactams by rapidly hydrolyzing the amide group of the beta-lactam ring. Specifically, the class A extended-spectrum beta-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a beta-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.

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pH-induced conformational change of the beta-barrel-forming protein OmpG reconstituted into native E. coli lipids.

Publication Date: 2010 Feb 26 PMID: 20036258
Authors: Mari, S. A. - Koster, S. - Bippes, C. A. - Yildiz, O. - Kuhlbrandt, W. - Muller, D. J.
Journal: J Mol Biol

A gating mechanism of the beta-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the beta-strands that form the transmembrane beta-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.

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Molecular and structural insight into proNGF engagement of p75NTR and sortilin.

Publication Date: 2010 Mar 5 PMID: 20036257
Authors: Feng, D. - Kim, T. - Ozkan, E. - Light, M. - Torkin, R. - Teng, K. K. - Hempstead, B. L. - Garcia, K. C.
Journal: J Mol Biol

Nerve growth factor (NGF) is initially synthesized as a precursor, proNGF, that is cleaved to release its C-terminal mature form. Recent studies suggested that proNGF is not an inactive precursor but acts as a signaling ligand distinct from its mature counterpart. proNGF and mature NGF initiate opposing biological responses by utilizing both distinct and shared receptor components. In this study, we carried out structural and biochemical characterization of proNGF interactions with p75NTR and sortilin. We crystallized proNGF complexed to p75NTR and present the structure at 3.75-A resolution. The structure reveals a 2:2 symmetric binding mode, as compared with the asymmetric structure of a previously reported crystal structure of mature NGF complexed to p75NTR and the 2:2 symmetric complex of neurotrophin-3 (NT-3) and p75NTR. Here, we discuss the possible origins and implications of the different stoichiometries. In the proNGF-p75NTR complex, the pro regions of proNGF are mostly disordered and two hairpin loops (loop 2) at the top of the NGF dimer have undergone conformational changes in comparison with mature NT structures, suggesting possible interactions with the propeptide. We further explored the binding characteristics of proNGF to sortilin using surface plasmon resonance and cell-based assays and determined that calcium ions promote the formation of a stable ternary complex of proNGF-sortilin-p75NTR. These results, together with those of previous structural and mechanistic studies of NT-receptor interactions, suggest the potential for distinct signaling activities through p75NTR mediated by different NT-induced conformational changes.

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Crystal structures of the CERT START domain with inhibitors provide insights into the mechanism of ceramide transfer.

Publication Date: 2010 Feb 19 PMID: 20036255
Authors: Kudo, N. - Kumagai, K. - Matsubara, R. - Kobayashi, S. - Hanada, K. - Wakatsuki, S. - Kato, R.
Journal: J Mol Biol

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Omega1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Omega1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.

MeSH Categories: Amides/chemistry/metabolism, Animals, Biological Transport/physiology, CHO Cells, Ceramides/*metabolism, Cricetinae, Cricetulus, Crystallography, X-Ray, Humans, Models, Molecular, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs, Protein Kinase Inhibitors/*chemistry/*metabolism, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases/antagonists &, inhibitors/*chemistry/*metabolism, Signal Transduction/physiology

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Evolutionary optimization of computationally designed enzymes: Kemp eliminases of the KE07 series.

Publication Date: 2010 Mar 5 PMID: 20036254
Authors: Khersonsky, O. - Rothlisberger, D. - Dym, O. - Albeck, S. - Jackson, C. J. - Baker, D. - Tawfik, D. S.
Journal: J Mol Biol

Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K(M) value of approximately 2600 s(-1) M(-1) and an approximately 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.

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Crystal structure of ArgP from Mycobacterium tuberculosis confirms two distinct conformations of full-length LysR transcriptional regulators and reveals its function in DNA binding and transcriptional regulation.

Publication Date: 2010 Mar 5 PMID: 20036253
Authors: Zhou, X. - Lou, Z. - Fu, S. - Yang, A. - Shen, H. - Li, Z. - Feng, Y. - Bartlam, M. - Wang, H. - Rao, Z.
Journal: J Mol Biol

Mycobacterium tuberculosis presents a challenging medical problem partly due to its persistent nonreplicative state. The inhibitor of chromosomal replication (iciA) protein encoded by M. tuberculosis has been suggested to inhibit chromosome replication initiation in vitro. However, iciA has also been identified as arginine permease (ArgP), a regulatory transcription factor for arginine outward transport. In order to understand the function of ArgP, we have determined its crystal structure by X-ray crystallography to a resolution of 2.7 A. ArgP is a member of the LysR-type transcriptional regulators (LTTRs) and forms a homodimer with each subunit containing two domains: a DNA binding domain (DBD) and a regulatory domain (RD). Two conformationally distinct subunits were identified: closed subunit and open subunit. This phenomenon was first observed in LTTR CbnR, but not in LTTR CrgA, and might be common in LTTRs. We identified two forms of dimers: DBD-type dimers and RD-type dimers. The former is confirmed in solution, and the latter is considered to form oligomers during function. We provide the first structural insights into the interaction of the extreme C-terminal residues with the DBD, which is confirmed by mutagenesis and analytical ultracentrifugation to be important for stability of the functional dimer. The structure serves as a model to suggest how three critical aspects, namely, DNA binding, homo-oligomerization, and interaction with RNAP, are mediated during regulation processing. A model is proposed for the LysR family of dimeric regulators.

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Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family.

Publication Date: 2010 Mar 19 PMID: 20036252
Authors: Kerff, F. - Petrella, S. - Mercier, F. - Sauvage, E. - Herman, R. - Pennartz, A. - Zervosen, A. - Luxen, A. - Frere, J. M. - Joris, B. - Charlier, P.
Journal: J Mol Biol

AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-gamma-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-gamma-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.

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N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.

Publication Date: 2010 Mar 5 PMID: 20036251
Authors: Brannigan, J. A. - Smith, B. A. - Yu, Z. - Brzozowski, A. M. - Hodgkinson, M. R. - Maroof, A. - Price, H. P. - Meier, F. - Leatherbarrow, R. J. - Tate, E. W. - Smith, D. F. - Wilkinson, A. J.
Journal: J Mol Biol

N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovani NMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 A resolution. The structure has as its defining feature a 14-stranded twisted beta-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.

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Combining EPR with fluorescence spectroscopy to monitor conformational changes at the myosin nucleotide pocket.

Publication Date: 2010 Mar 5 PMID: 20036250
Authors: Naber, N. - Malnasi-Csizmadia, A. - Purcell, T. J. - Cooke, R. - Pate, E.
Journal: J Mol Biol

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WTSLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin myosin diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins.

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Crystal structure of a thermophilic GrpE protein: insight into thermosensing function for the DnaK chaperone system.

Publication Date: 2010 Mar 5 PMID: 20036249
Authors: Nakamura, A. - Takumi, K. - Miki, K.
Journal: J Mol Biol

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE(Tth)) at 3.23 A resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE(Eco)), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE(Tth). In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE(Eco) structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE(Tth) correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE(Tth) causes functional deficiency of nucleotide exchange factor in response to the heat shock.

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Evolutionary trace annotation of protein function in the structural proteome.

Publication Date: 2010 Mar 12 PMID: 20036248
Authors: Erdin, S. - Ward, R. M. - Venner, E. - Lichtarge, O.
Journal: J Mol Biol

By design, structural genomics (SG) solves many structures that cannot be assigned function based on homology to known proteins. Alternative function annotation methods are therefore needed and this study focuses on function prediction with three-dimensional (3D) templates: small structural motifs built of just a few functionally critical residues. Although experimentally proven functional residues are scarce, we show here that Evolutionary Trace (ET) rankings of residue importance are sufficient to build 3D templates, match them, and then assign Gene Ontology (GO) functions in enzymes and non-enzymes alike. In a high-specificity mode, this Evolutionary Trace Annotation (ETA) method covered half (53%) of the 2384 annotated SG protein controls. Three-quarters (76%) of predictions were both correct and complete. The positive predictive value for all GO depths (all-depth PPV) was 84%, and it rose to 94% over GO depths 1-3 (depth 3 PPV). In a high-sensitivity mode, coverage rose significantly (84%), while accuracy fell moderately: 68% of predictions were both correct and complete, all-depth PPV was 75%, and depth 3 PPV was 86%. These data concur with prior mutational experiments showing that ET rank information identifies key functional determinants in proteins. In practice, ETA predicted functions in 42% of 3461 unannotated SG proteins. In 529 cases--including 280 non-enzymes and 21 for metal ion ligands--the expected accuracy is 84% at any GO depth and 94% down to GO depth 3, while for the remaining 931 the expected accuracies are 60% and 71%, respectively. Thus, local structural comparisons of evolutionarily important residues can help decipher protein functions to known reliability levels and without prior assumption on functional mechanisms. ETA is available at http://mammoth.bcm.tmc.edu/eta.

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Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of alpha-helical outer membrane proteins.

Publication Date: 2010 Feb 26 PMID: 20026336
Authors: Thornton, N. - Stroud, D. A. - Milenkovic, D. - Guiard, B. - Pfanner, N. - Becker, T.
Journal: J Mol Biol

The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.

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Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

Publication Date: 2010 Mar 5 PMID: 20026132
Authors: Banroques, J. - Doere, M. - Dreyfus, M. - Linder, P. - Tanner, N. K.
Journal: J Mol Biol

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.

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Role of the adenine ligand on the stabilization of the secondary and tertiary interactions in the adenine riboswitch.

Publication Date: 2010 Mar 12 PMID: 20026131
Authors: Priyakumar, U. D. - MacKerell, A. D. Jr
Journal: J Mol Biol

Riboswitches are RNA-based genetic control elements that function via a conformational transition mechanism when a specific target molecule binds to its binding pocket. To facilitate an atomic detail interpretation of experimental investigations on the role of the adenine ligand on the conformational properties and kinetics of folding of the add adenine riboswitch, we performed molecular dynamics simulations in both the presence and the absence of the ligand. In the absence of ligand, structural deviations were observed in the J23 junction and the P1 stem. Destabilization of the P1 stem in the absence of ligand involves the loss of direct stabilizing interactions with the ligand, with additional contributions from the J23 junction region. The J23 junction of the riboswitch is found to be more flexible, and the tertiary contacts among the junction regions are altered in the absence of the adenine ligand; results suggest that the adenine ligand associates and dissociates from the riboswitch in the vicinity of J23. Good agreement was obtained with the experimental data with the results indicating dynamic behavior of the adenine ligand on the nanosecond time scale to be associated with the dynamic behavior of hydrogen bonding with the riboswitch. Results also predict that direct interactions of the adenine ligand with U74 of the riboswitch are not essential for stable binding although it is crucial for its recognition. The possibility of methodological artifacts and force-field inaccuracies impacting the present observations was checked by additional molecular dynamics simulations in the presence of 2,6-diaminopurine and in the crystal environment.

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Intermonomer hydrogen bonds enhance GxxxG-driven dimerization of the BNIP3 transmembrane domain: roles for sequence context in helix-helix association in membranes.

Publication Date: 2010 Mar 5 PMID: 20026130
Authors: Lawrie, C. M. - Sulistijo, E. S. - MacKenzie, K. R.
Journal: J Mol Biol

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.

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The recombinant amyloid-beta peptide Abeta1-42 aggregates faster and is more neurotoxic than synthetic Abeta1-42.

Publication Date: 2010 Feb 12 PMID: 20026079
Authors: Finder, V. H. - Vodopivec, I. - Nitsch, R. M. - Glockshuber, R.
Journal: J Mol Biol

Aggregation of the amyloid-beta (Abeta) peptide is considered a central event in the pathogenesis of Alzheimer's disease (AD). In order to bypass methodological bias related to a variety of impurities commonly present in typical preparations of synthetic Abeta, we developed a simple, generally applicable method for recombinant production of human Abeta and Abeta variants in Escherichia coli that provides milligram quantities of Abeta in very high purity and yield. Amyloid fibril formation in vitro by human Abeta1-42, the key amyloidogenic Abeta species in AD, was completed threefold faster with recombinant Abeta1-42 compared to synthetic preparations. In addition, recombinant Abeta1-42 was significantly more toxic to cultured rat primary cortical neurons, and it was more toxic in vivo, as shown by strongly increased induction of abnormal phosphorylation of tau and tau aggregation into neurofibrillary tangles in brains of P301L tau transgenic mice. We conclude that even small amounts of impurities in synthetic Abeta-including a significant fraction of racemized peptides that cannot be avoided due to the technical limitations of peptide synthesis--prevent or slow Abeta incorporation into the regular quaternary structure of growing beta-amyloid fibrils. The results validate the use of recombinant Abeta1-42 for both in vitro and in vivo studies addressing the mechanisms underlying Abeta aggregation and its related biological consequences for the pathophysiology, therapy, and prevention of AD.

MeSH Categories: Amino Acid Sequence, Amyloid/chemistry, Amyloid beta-Protein/*chemistry/isolation &, purification/*toxicity/ultrastructure, Animals, Cells, Cultured, Humans, Mice, Molecular Sequence Data, Neurons/*drug effects, Neurotoxins/chemistry/*toxicity, Peptide Fragments/*chemistry/isolation &, purification/*toxicity/ultrastructure, Protein Structure, Quaternary, Rats, Recombinant Proteins/*chemistry/isolation & purification/*toxicity, tau Proteins/metabolism

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Binding of S-methyl-5'-thioadenosine and S-adenosyl-L-methionine to protein MJ0100 triggers an open-to-closed conformational change in its CBS motif pair.

Publication Date: 2010 Feb 26 PMID: 20026078
Authors: Lucas, M. - Encinar, J. A. - Arribas, E. A. - Oyenarte, I. - Garcia, I. G. - Kortazar, D. - Fernandez, J. A. - Mato, J. M. - Martinez-Chantar, M. L. - Martinez-Cruz, L. A.
Journal: J Mol Biol

Cystathionine beta-synthase (CBS) domains are small motifs that are present in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The protein MJ0100 from Methanocaldococcus jannaschii includes a DUF39 domain of so far unknown function and a CBS domain pair (Bateman domain) at its C-terminus. This work presents the crystallographic analysis of four different states of the CBS motif pair of MJ0100 in complex with different numbers of S-adenosyl-L-methionine (SAM) and S-methyl-5'-thioadenosine (MTA) ligands, providing evidence that ligand-induced conformational reorganization of Bateman domain dimers could be an important regulatory mechanism. These observations are in contrast to what is known from most of the other Bateman domain structures but are supported by recent studies on the magnesium transporter MgtE. Our structures represent the first example of a CBS domain protein complexed with SAM and/or MTA and might provide a structural basis for understanding the molecular mechanisms regulated by SAM upon binding to the C-terminal domain of human CBS, whose structure remains unknown.

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Conversion of Abeta42 into a folded soluble native-like protein using a semi-random library of amphipathic helices.

Publication Date: 2010 Mar 12 PMID: 20026077
Authors: Arslan, P. E. - Mulligan, V. K. - Ho, S. - Chakrabartty, A.
Journal: J Mol Biol

The amyloid cascade model hypothesizes that neurotoxic oligomers or aggregates formed by the Alzheimer amyloid peptide (Abeta) cause disease pathology in Alzheimer's disease. Attempted treatment strategies for Alzheimer's disease have involved either inhibiting Abeta oligomerization or aggregation, or dissolving existing aggregates. Blocking such downhill processes, however, has proved daunting. We have used a different approach that targets Abeta before the oligomerization cascade begins. We predicted that an amphipathic helix could convert Abeta into a native-like protein and inhibit initiation of oligomerization and aggregation. This idea was tested with a designed library and genetic screen. We exhaustively screened a library of semi-randomized amphipathic helical sequences, each expressed as a fusion protein with an Abeta42-yellow fluorescent protein sequence serving as a reporter for folding and solubilization. This yielded an amphipathic helix capable of initiating native-like folding in Abeta42 and preventing aggregation. This amphipathic helix has direct application to Alzheimer's disease therapy development.

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Accessory alpha-helix of complexin I can displace VAMP2 locally in the complexin-SNARE quaternary complex.

Publication Date: 2010 Feb 26 PMID: 20026076
Authors: Lu, B. - Song, S. - Shin, Y. K.
Journal: J Mol Biol

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory alpha-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory alpha-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory alpha-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.

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Structure of the adenylylation domain of E. coli glutamine synthetase adenylyl transferase: evidence for gene duplication and evolution of a new active site.

Publication Date: 2010 Feb 26 PMID: 20026075
Authors: Xu, Y. - Carr, P. D. - Vasudevan, S. G. - Ollis, D. L.
Journal: J Mol Biol

The X-ray structure of the C-terminal fragment, containing residues 449-946, of Escherichia coli glutamine synthetase adenylyl transferase (ATase) has been determined. ATase is part of the cascade that regulates the enzymatic activity of E. coli glutamine synthetase, a key component of the cell's machinery for the uptake of ammonia. It has two enzymatic activities, adenylyl removase (AR) and adenylyl transferase (AT), which are located in distinct catalytic domains that are separated by a regulatory (R) domain. We previously reported the three-dimensional structure of the AR domain (residues 1-440). The present structure contains both the R and AT domains. AR and AT share 24% sequence identity and also contain the beta-polymerase motif that is characteristic of many nucleotidylyl transferase enzymes. The structures overlap with an rmsd of 2.4 A when the superhelical R domain is omitted. A model for the complete ATase molecule is proposed, along with some refinements of domain boundaries. A rather more speculative model for the complex of ATase with glutamine synthetase and the nitrogen signal transduction protein PII is also presented.

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The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, "as-isolated" state.

Publication Date: 2010 Mar 5 PMID: 20026074
Authors: Marques, M. C. - Coelho, R. - De Lacey, A. L. - Pereira, I. A. - Matias, P. M.
Journal: J Mol Biol

Hydrogen is a good energy vector, and its production from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biological production of hydrogen because they are capable of high production rates even in the presence of moderate amounts of O(2), lessening the requirements for anaerobic conditions. The three-dimensional structure of the [NiFeSe] Hase from Desulfovibrio vulgaris Hildenborough has been determined in its oxidised "as-isolated" form at 2.04-A resolution. Remarkably, this is the first structure of an oxidised Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra sulfur atom is observed binding Ni and Se, leading to a SeCys conformation that shields the NiFe site from contact with oxygen. This structure provides several insights that may explain the fast activation and O(2) tolerance of these enzymes.

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Modulation of an active-site cysteine pKa allows PDI to act as a catalyst of both disulfide bond formation and isomerization.

Publication Date: 2010 Mar 5 PMID: 20026073
Authors: Karala, A. R. - Lappi, A. K. - Ruddock, L. W.
Journal: J Mol Biol

Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond reduction and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pK(a) values of the active-site cysteines of PDI. Here, we examined the role of arginine 120 in modulating the pK(a) values of these cysteines. We find that arginine 120 plays a significant role in modulating the pK(a) of the C-terminal active-site cysteine in the a domain of PDI and plays a role in determining the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pK(a). Mutation of arginine 120 and the corresponding residue, arginine 461, in the a' domain severely reduces the ability of PDI to catalyze disulfide bond formation and reduction but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pK(a) of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidation and isomerization reactions.

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Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes.

Publication Date: 2010 Mar 5 PMID: 20026072
Authors: Tylichova, M. - Kopecny, D. - Morera, S. - Briozzo, P. - Lenobel, R. - Snegaroff, J. - Sebela, M.
Journal: J Mol Biol

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived omega-aminoaldehydes to the corresponding omega-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with beta-nicotinamide adenine dinucleotide (NAD(+)) at 2.4 and 2.15 A resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD(+) as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD(+) binding site. While the NAD(+) binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into gamma-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, beta-alanine betaine and gamma-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.

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Modulation of polyglutamine conformations and dimer formation by the N-terminus of huntingtin.

Publication Date: 2010 Mar 12 PMID: 20026071
Authors: Williamson, T. E. - Vitalis, A. - Crick, S. L. - Pappu, R. V.
Journal: J Mol Biol

Polyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins in the intrinsic conformational and aggregation properties of polyglutamine. We report results from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q(N) constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine-containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation and identify roles for flanking sequences in the modulation of polyglutamine aggregation.

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Functional analysis of human tRNA isodecoders.

Publication Date: 2010 Feb 26 PMID: 20026070
Authors: Geslain, R. - Pan, T.
Journal: J Mol Biol

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families. Whether sequence diversity among isodecoder tRNA genes reflects functional variability is an open question. To address this, we developed a method to quantify the efficiency of tRNA isodecoders in stop-codon suppression in human cell lines. First, a green fluorescent protein (GFP) gene that contains a single UAG stop codon at two distinct locations is introduced. GFP is only produced when a tRNA suppressor containing CUA anticodon is co-transfected with the GFP gene. The suppression efficiency is examined for 31 tRNA isodecoders (all contain CUA anticodon), 21 derived from four isoacceptor families of tRNASer genes, 7 from five families of tRNALeu genes, and 3 from three families of tRNAAla genes. We found that isodecoder tRNAs display a large difference in their suppression efficiency. Among those with above background suppression activity, differences of up to 20-fold were observed. We were able to tune tRNA suppression efficiency by subtly adjusting the tRNA sequence and inter-convert poor suppressors into potent ones. We also demonstrate that isodecoder tRNAs with varying suppression efficiencies have similar stability and exhibit similar levels of aminoacylation in vivo. Our results indicate that naturally occurring tRNA isodecoders can have large functional variations and suggest that some tRNA isodecoders may perform a function distinct from translation.

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Crystal structure of the LasA virulence factor from Pseudomonas aeruginosa: substrate specificity and mechanism of M23 metallopeptidases.

Publication Date: 2010 Mar 5 PMID: 20026068
Authors: Spencer, J. - Murphy, L. M. - Conners, R. - Sessions, R. B. - Gamblin, S. J.
Journal: J Mol Biol

Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.

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The prototypical H+/galactose symporter GalP assembles into functional trimers.

Publication Date: 2010 Feb 26 PMID: 20006622
Authors: Zheng, H. - Taraska, J. - Merz, A. J. - Gonen, T.
Journal: J Mol Biol

Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.

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Discrimination between closely related cellular metabolites by the SAM-I riboswitch.

Publication Date: 2010 Feb 26 PMID: 20006621
Authors: Montange, R. K. - Mondragon, E. - van Tyne, D. - Garst, A. D. - Ceres, P. - Batey, R. T.
Journal: J Mol Biol

The SAM-I riboswitch is a cis-acting element of genetic control found in bacterial mRNAs that specifically binds S-adenosylmethionine (SAM). We previously determined the 2.9-A X-ray crystal structure of the effector-binding domain of this RNA element, revealing details of RNA-ligand recognition. To improve this structure, variations were made to the RNA sequence to alter lattice contacts, resulting in a 0.5-A improvement in crystallographic resolution and allowing for a more accurate refinement of the crystallographic model. The basis for SAM specificity was addressed by a structural analysis of the RNA complexed to S-adenosylhomocysteine (SAH) and sinefungin and by measuring the affinity of SAM and SAH for a series of mutants using isothermal titration calorimetry. These data illustrate the importance of two universally conserved base pairs in the RNA that form electrostatic interactions with the positively charged sulfonium group of SAM, thereby providing a basis for discrimination between SAM and SAH.

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Crystal structure analysis of free and substrate-bound 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans.

Publication Date: 2010 Feb 26 PMID: 20006620
Authors: Kachalova, G. S. - Bourenkov, G. P. - Mengesdorf, T. - Schenk, S. - Maun, H. R. - Burghammer, M. - Riekel, C. - Decker, K. - Bartunik, H. D.
Journal: J Mol Biol

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.

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Characterisation of a GroEL single-ring mutant that supports growth of Escherichia coli and Has GroES-dependent ATPase activity.

Publication Date: 2010 Mar 12 PMID: 20006619
Authors: Kovacs, E. - Sun, Z. - Liu, H. - Scott, D. J. - Karsisiotis, A. I. - Clarke, A. R. - Burston, S. G. - Lund, P. A.
Journal: J Mol Biol

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.

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Molecular determinants required for selective interactions between the thyroid hormone receptor homodimer and the nuclear receptor corepressor N-CoR.

Publication Date: 2010 Feb 26 PMID: 20006618
Authors: Kim, J. Y. - Son, Y. L. - Kim, J. S. - Lee, Y. C.
Journal: J Mol Biol

The unliganded nuclear receptor (NR) generally recruits the NR corepressor (N-CoR) and the silencing mediator of retinoid and thyroid hormone receptor via its direct binding to the extended helical motif within dual NR-interaction domains (IDs) of corepressors. Interestingly, N-CoR has a third ID (ID3) upstream of two IDs (ID1 and ID2) and its core motif (IDVII), rather than an extended helical motif, is known to be involved directly in the exclusive interaction of ID3 with the thyroid hormone receptor (TR). Here, we investigated the molecular determinants of the TR interaction with ID3 to understand the molecular basis of the N-CoR preference shown by the TR homodimer. Using a one- plus two-hybrid system, we identified the specific residues of N-CoR-ID2 and N-CoR-ID3 that are required for stable association of N-CoR with the TR homodimer. By swapping experiments and mutagenesis studies, we found that the C-terminally flanked residues of the core motif of ID3 contribute to the TR preference for N-CoR-ID3, suggesting that an extended three-turn helix might form within the ID3 via a C-terminal extension (IDVIITRQI) and participate directly in the TR-specific interaction. Structural modeling of the ID3 motif on TR-LBD is consistent with this conclusion. Notably, we identified a novel interaction between N-CoR-ID3 and orphan NR RevErb that is mediated by the residues crucial also in TR binding. These observations raise the intriguing possibility that NR homodimers such as TR and RevErb display preferential binding to the N-CoR corepressor via their specific interactions with ID3, which is normally absent from the silencing mediator of retinoid and thyroid hormone receptor.

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The electrostatics of VDAC: implications for selectivity and gating.

Publication Date: 2010 Feb 26 PMID: 20005234
Authors: Choudhary, O. P. - Ujwal, R. - Kowallis, W. - Coalson, R. - Abramson, J. - Grabe, M.
Journal: J Mol Biol

The voltage-dependent anion channel (VDAC) is the major pathway mediating the transfer of metabolites and ions across the mitochondrial outer membrane. Two hallmarks of the channel in the open state are high metabolite flux and anion selectivity, while the partially closed state blocks metabolites and is cation selective. Here we report the results from electrostatics calculations carried out on the recently determined high-resolution structure of murine VDAC1 (mVDAC1). Poisson-Boltzmann calculations show that the ion transfer free energy through the channel is favorable for anions, suggesting that mVDAC1 represents the open state. This claim is buttressed by Poisson-Nernst-Planck calculations that predict a high single-channel conductance indicative of the open state and an anion selectivity of 1.75--nearly a twofold selectivity for anions over cations. These calculations were repeated on mutant channels and gave selectivity changes in accord with experimental observations. We were then able to engineer an in silico mutant channel with three point mutations that converted mVDAC1 into a channel with a preference for cations. Finally, we investigated two proposals for how the channel gates between the open and the closed state. Both models involve the movement of the N-terminal helix, but neither motion produced the observed voltage sensitivity, nor did either model result in a cation-selective channel, which is observed experimentally. Thus, we were able to rule out certain models for channel gating, but the true motion has yet to be determined.

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Lineage-specific differences in the amino acid substitution process.

Publication Date: 2010 Mar 12 PMID: 20004669
Authors: Huzurbazar, S. - Kolesov, G. - Massey, S. E. - Harris, K. C. - Churbanov, A. - Liberles, D. A.
Journal: J Mol Biol

In Darwinian evolution, mutations occur approximately at random in a gene, turned into amino acid mutations by the genetic code. Some mutations are fixed to become substitutions and some are eliminated from the population. Partitioning pairs of closely related species with complete genome sequences by average population size of each pair, we looked at the substitution matrices generated for these partitions and compared the substitution patterns between species. We estimated a population genetic model that relates the relative fixation probabilities of different types of mutations to the selective pressure and population size. Parameterizations of the average and distribution of selective pressures for different amino acid substitution types in different population size comparisons were generated with a Bayesian framework. We found that partitions in population size as well as in substitution type are required to explain the substitution data. Selection coefficients were found to decrease with increasingly radical amino acid substitution and with increasing effective population size. To further explore the role of underlying processes in amino acid substitution, we analyzed embryophyte (plant) gene families from TAED (The Adaptive Evolution Database), where solved structures for at least one member exist in the Protein Data Bank. Using PAML, we assigned branches to three categories: strong negative selection, moderate negative selection/neutrality, and positive diversifying selection. Focusing on the first and third categories, we identified sites changing along gene family lineages and observed the spatial patterns of substitution. Selective sweeps were expected to create primary sequence clustering under positive diversifying selection. Co-evolution through direct physical interaction was expected to cause tertiary structural clustering. Under both positive and negative selection, the substitution patterns were found to be nonrandom. Under positive diversifying selection, significant independent signals were found for primary and tertiary sequence clustering, suggesting roles for both selective sweeps and direct physical interaction. Under strong negative selection, the signals were not found to be independent. All together, a complex interplay of population genetic and protein thermodynamics forces is suggested.

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The outer membrane usher guarantees the formation of functional pili by selectively catalyzing donor-strand exchange between subunits that are adjacent in the mature pilus.

Publication Date: 2010 Feb 12 PMID: 20004668
Authors: Nishiyama, M. - Glockshuber, R.
Journal: J Mol Biol

Type 1 pili from uropathogenic Escherichia coli are a prototype of adhesive surface organelles assembled and secreted by the conserved chaperone/usher pathway. They are composed of four different homologous protein subunits that need to be assembled in a defined order. In the periplasm, the pilus chaperone FimC donates a beta-strand segment to the subunits to complete their imperfect immunoglobulin-like fold. During subunit assembly, this segment of the chaperone is displaced by an amino-terminal extension of an incoming subunit in a reaction termed donor-strand exchange. To date, the molecular mechanisms underlying the coordinated subunit assembly, in particular the role of the outer membrane usher FimD, are still poorly understood. Here we show that the binding of complexes between FimC and the different pilus subunits to the amino-terminal substrate recognition domain of FimD is an extremely fast process, with association rate constants in the range of 10(7)-10(8) M(-)(1) s(-1) at 20 degrees C. Furthermore, we demonstrate that the ordered assembly of pilus subunits is a consequence of the usher's ability to selectively catalyze the assembly of defined subunit-subunit pairs that are adjacent in the mature pilus. The usher therefore coordinates the assembly of pilus subunits at the stage of donor-strand exchange between pairs of subunits and not at the level of the initial binding of chaperone-subunit complexes.

MeSH Categories: *Biocatalysis, Cell Membrane/*metabolism, Escherichia coli/metabolism, Escherichia coli Proteins/chemistry/metabolism, Fimbriae, Bacterial/*metabolism, Fluorescein/metabolism, Fluorescence, Kinetics, Molecular Chaperones/chemistry/*metabolism, Protein Binding, Protein Structure, Secondary, Protein Subunits/*metabolism, Staining and Labeling, Temperature, Titrimetry

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The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris.

Publication Date: 2010 Feb 26 PMID: 20004667
Authors: Chin, K. H. - Lee, Y. C. - Tu, Z. L. - Chen, C. H. - Tseng, Y. H. - Yang, J. M. - Ryan, R. P. - McCarthy, Y. - Dow, J. M. - Wang, A. H. - Chou, S. H.
Journal: J Mol Biol

Cyclic-di-GMP [bis-(3'-5')-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with micromolar affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.

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Identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands.

Publication Date: 2010 Feb 26 PMID: 20004209
Authors: Coombs, P. J. - Harrison, R. - Pemberton, S. - Quintero-Martinez, A. - Parry, S. - Haslam, S. M. - Dell, A. - Taylor, M. E. - Drickamer, K.
Journal: J Mol Biol

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity. Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.

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Characterization of the head-to-tail overlap complexes formed by human lamin A, B1 and B2 "half-minilamin" dimers.

Publication Date: 2010 Feb 26 PMID: 20004208
Authors: Kapinos, L. E. - Schumacher, J. - Mucke, N. - Machaidze, G. - Burkhard, P. - Aebi, U. - Strelkov, S. V. - Herrmann, H.
Journal: J Mol Biol

Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and s-values (1.6-1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher s-values (2.3-2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a "combinatorial" head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.

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Light activation of rhodopsin: insights from molecular dynamics simulations guided by solid-state NMR distance restraints.

Publication Date: 2010 Feb 26 PMID: 20004206
Authors: Hornak, V. - Ahuja, S. - Eilers, M. - Goncalves, J. A. - Sheves, M. - Reeves, P. J. - Smith, S. O.
Journal: J Mol Biol

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (>1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from 13C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.

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Increase in backbone mobility of the VTS1p-SAM domain on binding to SRE-RNA.

Publication Date: 2010 Feb 26 PMID: 20004205
Authors: Ravindranathan, S. - Oberstrass, F. C. - Allain, F. H.
Journal: J Mol Biol

The sterile alpha motif (SAM) domain of VTS1p, a posttranscriptional gene regulator, belongs to a family of SAM domains conserved from yeast to humans. Even though SAM domains were originally classified as protein-protein interaction domains, recently, it was shown that the yeast VTS1p-SAM and the SAM domain of its Drosophila homolog Smaug can specifically recognize RNA hairpins termed Smaug recognition element (SRE). Structural studies of the SRE-RNA complex of VTS1p-SAM revealed that the SAM domain primarily recognizes the shape of the RNA fold induced by the Watson-Crick base-pairing in the RNA pentaloop. Only the central G nucleotide is specifically recognized. The VTS1p-SAM domain recognizes SRE-RNAs with a CNGGN pentaloop where N is any nucleotide. The C1-G4 base pair in the wild type can be replaced by any pair of nucleotides that can form base pairs even though the binding affinity is greatest with a pyrimidine in position 1 and a purine in position 4. The interaction thus combines elements of sequence-specific and non-sequence-specific recognitions. The lack of structural rearrangements in either partner following binding is rather intriguing, suggesting that molecular dynamics may play an important role in imparting relaxed specificity with respect to the exact combination of nucleotides in the loop, except for the central nucleotide. In this work, we extend our previous studies of SRE-RNA interaction with VTS1p, by comparing the dynamics of the VTS1p-SAM domain both in its free form and when bound to SRE-RNA. The 15N relaxation studies of backbone dynamics suggest the presence of a dynamic interaction interface, with residues associated with specific G3 recognition becoming more rigid on RNA binding while other regions attain increased flexibility. The results parallel the observations from our studies of dynamics changes in SRE-RNA upon binding to VTS1p-SAM and shows that molecular dynamics could play a crucial role in modulating binding affinity and possibly contribute to the free energy of the interaction through an entropy-driven mechanism.

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PCP consensus sequences of flaviviruses: correlating variance with vector competence and disease phenotype.

Publication Date: 2010 Feb 26 PMID: 19969003
Authors: Danecek, P. - Lu, W. - Schein, C. H.
Journal: J Mol Biol

BACKGROUND: Computational methods are needed to design multivalent vaccines against flaviviruses (FVs) such as the West Nile virus or the dengue virus (DENV). OBJECTIVE: We aimed to use physicochemical property (PCP) consensus sequences of FV strains to delineate conserved motifs, areas of maximum variability, and specific loci that correlate with arthropod vector, serotype, and disease severity. METHODS: PCP consensus sequences for 27 species were prepared from 928 annotated sequences catalogued in Flavitrack. Alignments of these correlated well with the known structures of the NS3 protease domain and envelope (E) proteins. The PCPMer suite was used to identify motifs common to all FVs. Areas of PCP variability that correlated with phenotype were plotted on the structures. RESULTS: Despite considerable diversity at the amino acid level, PCPs for both proteins were well conserved throughout the FVs. A series of insertions in E separated tick- from mosquito-borne viruses and all arthropod-borne viruses from isolates with no known vector or directly from insects. Comparison of a PCP consensus sequence of E derived from 600 DENV strains (DENV600) with individual ones for DENV1-DENV4 showed that most major serotype-specific variation occurs near these insertions. The DENV600 differed from one prepared from eight hemorrhagic or fatal strains from four DENV serotypes at only three positions, two of which overlap known escape mutant sites. CONCLUSIONS: Comparing consensus sequences showed that substantial changes occur in only a few areas of the E protein. PCP consensus sequences can contribute to the design of multivalent vaccines.

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c/EBPbeta is a major regulatory element driving transcriptional activation of the CXCL12 promoter.

Publication Date: 2010 Feb 26 PMID: 19962993
Authors: Calonge, E. - Alonso-Lobo, J. M. - Escandon, C. - Gonzalez, N. - Bermejo, M. - Santiago, B. - Mestre, L. - Pablos, J. L. - Caruz, A. - Alcami, J.
Journal: J Mol Biol

CXCL12 is considered a constitutively expressed chemokine with homeostatic functions. However, induction of CXCL12 expression and its potential role in several pathologic conditions have been reported, suggesting that CXCL12 gene expression can be induced by different stimuli. To elucidate the molecular mechanisms involved in the regulation of CXCL12 gene expression, we aim to define the molecular factors that operate at the transcriptional level. Basal, constitutive expression of CXCL12 was dependent on basic helix-loop-helix factors. Transcriptional up-regulation of the CXCL12 gene was induced by cellular confluence or inflammatory stimuli such as interleukin-1 and interleukin-6, in a CCAAT/enhancer binding protein beta (c/EBPbeta)-dependent manner. Chromatin immunoprecipitation assays confirmed c/EBPbeta binding to a specific response element located at -1171 of the promoter region of CXCL12. Our data show that c/EBPbeta is a major regulatory element driving transcription of the CXCL12 gene in response to cytokines and cell confluence.

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Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex.

Publication Date: 2010 Feb 26 PMID: 19962990
Authors: Prochniewicz, E. - Chin, H. F. - Henn, A. - Hannemann, D. E. - Olivares, A. O. - Thomas, D. D. - De La Cruz, E. M.
Journal: J Mol Biol

We used transient phosphorescence anisotropy to detect the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin strongly bound to single-headed fragments of muscle myosin subfragment 1 (S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at substoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than with muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects: muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly and muscle myosin binds more slowly to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound erythrosin iodoacetamide: while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex are determined by the isoform of the bound myosin in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

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Structural basis for human monoglyceride lipase inhibition.

Publication Date: 2010 Feb 26 PMID: 19962385
Authors: Bertrand, T. - Auge, F. - Houtmann, J. - Rak, A. - Vallee, F. - Mikol, V. - Berne, P. F. - Michot, N. - Cheuret, D. - Hoornaert, C. - Mathieu, M.
Journal: J Mol Biol

Monoglyceride lipase (MGL) is a serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. 2-AG is an endogenous ligand of cannabinoid receptors, involved in various physiological processes in the brain. We present here the first crystal structure of human MGL in its apo form and in complex with the covalent inhibitor SAR629. MGL shares the classic fold of the alpha/beta hydrolase family but depicts an unusually large hydrophobic occluded tunnel with a highly flexible lid at its entry and the catalytic triad buried at its end. Structures reveal the configuration of the catalytic triad and the shape and nature of the binding site of 2-AG. The bound structure of SAR629 highlights the key interactions for productive binding with MGL. The shape of the tunnel suggests a high druggability of the protein and provides an attractive template for drug discovery.

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Site-directed mutagenesis on human cystathionine-gamma-lyase reveals insights into the modulation of H2S production.

Publication Date: 2010 Feb 26 PMID: 19961860
Authors: Huang, S. - Chua, J. H. - Yew, W. S. - Sivaraman, J. - Moore, P. K. - Tan, C. H. - Deng, L. W.
Journal: J Mol Biol

In recent years, increased interest has been directed towards hydrogen sulfide (H2S) as the third gasotransmitter and its role in various diseases. Cystathionine-gamma-lyase (CSE) is one of the enzymes responsible for the endogenous production of H2S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H2S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H2S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5'-phosphate (PLP) regeneration due to weakened pi-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of alpha,gamma-elimination or alpha,beta-elimination reaction. Our study also shows that the rate of H2S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H2S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H2S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H2S-related diseases.

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Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis.

Publication Date: 2010 Feb 26 PMID: 19961859
Authors: Kouyama, T. - Kanada, S. - Takeguchi, Y. - Narusawa, A. - Murakami, M. - Ihara, K.
Journal: J Mol Biol

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-A resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic alpha-helix (A') at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.

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Ionic interactions promote transmembrane helix-helix association depending on sequence context.

Publication Date: 2010 Feb 19 PMID: 19961858
Authors: Herrmann, J. R. - Fuchs, A. - Panitz, J. C. - Eckert, T. - Unterreitmeier, S. - Frishman, D. - Langosch, D.
Journal: J Mol Biol

Folding and oligomerization of integral membrane proteins frequently depend on specific interactions of transmembrane helices. Interacting amino acids of helix-helix interfaces may form complex motifs and exert different types of molecular forces. Here, a set of strongly self-interacting transmembrane domains (TMDs), as isolated from a combinatorial library, was found to contain basic and acidic residues, in combination with polar nonionizable amino acids and C-terminal GxxxG motifs. Mutational analyses of selected sequences and reconstruction of high-affinity interfaces confirmed the cooperation of these residues in homotypic interactions. Probing heterotypic interaction indicated the presence of interhelical charge-charge interactions. Furthermore, simple motifs of an ionizable residue and GxxxG are significantly overrepresented in natural TMDs, and a specific combination of these motifs exhibits high-affinity heterotypic interaction. We conclude that intramembrane charge-charge interactions depend on sequence context. Moreover, they appear important for homotypic and heterotypic interactions of numerous natural TMDs.

MeSH Categories: Amino Acid Sequence/genetics/*physiology, Amino Acid Substitution/genetics/physiology, Consensus Sequence/genetics, Ions/*metabolism, Membrane Proteins/*chemistry/genetics/*metabolism, Mutagenesis, Site-Directed, Mutant Proteins/chemistry/metabolism, Protein Binding, Protein Interaction Domains and Motifs/genetics, Protein Interaction Mapping/methods, Protein Structure, Quaternary, Protein Structure, Secondary, Recombinant Proteins/analysis/chemistry/genetics/metabolism, Substrate Specificity/genetics

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Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism.

Publication Date: 2010 Feb 19 PMID: 19961857
Authors: Annalora, A. J. - Goodin, D. B. - Hong, W. X. - Zhang, Q. - Johnson, E. F. - Stout, C. D.
Journal: J Mol Biol

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 A resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A' and G', suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1alpha,25-dihydroxyvitamin D(3) binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K'', and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.

MeSH Categories: Amino Acid Sequence, Animals, Binding Sites, Crystallography, X-Ray, Cytochrome P-450 Enzyme System/chemistry/metabolism, Mitochondria/*enzymology, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Rats, Secosteroids/metabolism, Sequence Homology, Amino Acid, Steroid Hydroxylases/*chemistry/metabolism, Vitamin D/*metabolism

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Synergistic cooperation between two ClpB isoforms in aggregate reactivation.

Publication Date: 2010 Feb 26 PMID: 19961856
Authors: Nagy, M. - Guenther, I. - Akoyev, V. - Barnett, M. E. - Zavodszky, M. I. - Kedzierska-Mieszkowska, S. - Zolkiewski, M.
Journal: J Mol Biol

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

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Crystal structures of penicillin-binding proteins 4 and 5 from Haemophilus influenzae.

Publication Date: 2010 Feb 26 PMID: 19958776
Authors: Kawai, F. - Clarke, T. B. - Roper, D. I. - Han, G. J. - Hwang, K. Y. - Unzai, S. - Obayashi, E. - Park, S. Y. - Tame, J. R.
Journal: J Mol Biol

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel beta-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by beta-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.

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Searching DNA via a "Monkey Bar" mechanism: the significance of disordered tails.

Publication Date: 2010 Feb 26 PMID: 19958775
Authors: Vuzman, D. - Azia, A. - Levy, Y.
Journal: J Mol Biol

The search through nonspecific DNA for a specific site by proteins is known to be facilitated by sliding, hopping, and intersegment transfer between separate DNA strands, yet the driving forces of these protein dynamics from the molecular perspective are unclear. In this study, molecular features of the DNA search mechanism were explored for three homologous proteins (the HoxD9, Antp, and NK-2 homeodomains) using a simple computational model in which protein-DNA interactions are represented solely by electrostatic forces. In particular, we studied the impact that disordered N-terminal tails (N-tails), which are more common in DNA-binding proteins than in other proteins, have on the efficiency of DNA search. While the three homeodomain proteins were found to use similar binding interfaces in specific and nonspecific interactions with DNAs, their different electrostatic potentials affect the nature of their sliding dynamics. The different lengths and net charges of the N-tails of the homeodomains affect their motion along the DNA. The presence of an N-tail increases sliding propensity but slows linear diffusion along the DNA. When the search is performed in the presence of two parallel DNA molecules, a direct transfer, which is facilitated by the protein tail, from one nonspecific DNA to another occurs. The tailed proteins jump between two DNA molecules through an intermediate in which the recognition helix of the protein is adsorbed to one DNA fragment and the N-tail is adsorbed to the second, suggesting a "monkey bar" mechanism. Our study illustrates how the molecular architecture of proteins controls the efficiency of DNA scanning.

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Multiple novel classes of APRIL-specific receptor-blocking peptides isolated by phage display.

Publication Date: 2010 Feb 12 PMID: 19945466
Authors: Gordon, N. C. - Lien, S. - Johnson, J. - Wallweber, H. J. - Tran, T. - Currell, B. - Mathieu, M. - Quan, C. - Starovasnik, M. A. - Hymowitz, S. G. - Kelley, R. F.
Journal: J Mol Biol

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC(50) values of 0.49-27 microM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.

MeSH Categories: Alanine/metabolism, Amino Acid Sequence, Animals, B-Cell Maturation Antigen/chemistry/metabolism, Immobilized Proteins/metabolism, Mice, Models, Molecular, Molecular Sequence Data, Mutagenesis/genetics, *Peptide Library, Peptides/chemistry/*classification/*isolation & purification, Protein Binding, Protein Structure, Secondary, Solubility, Tumor Necrosis Factor Ligand Superfamily Member 13/*antagonists &, inhibitors/chemistry/metabolism

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Redox-dependent domain rearrangement of protein disulfide isomerase coupled with exposure of its substrate-binding hydrophobic surface.

Publication Date: 2010 Feb 19 PMID: 19944705
Authors: Serve, O. - Kamiya, Y. - Maeno, A. - Nakano, M. - Murakami, C. - Sasakawa, H. - Yamaguchi, Y. - Harada, T. - Kurimoto, E. - Yagi-Utsumi, M. - Iguchi, T. - Inaba, K. - Kikuchi, J. - Asami, O. - Kajino, T. - Oka, T. - Nakasako, M. - Kato, K.
Journal: J Mol Biol

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b' and a' domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a' domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a' domain influences the dynamic properties of the b' domain. Moreover, the SAXS profiles revealed that oxidation of the a' active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a' domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a "closed" form releasing the oxidized substrate.

MeSH Categories: Ascomycota/enzymology/metabolism, Binding Sites, Crystallography, X-Ray, *Hydrophobicity, Models, Biological, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Protein Disulfide-Isomerases/*chemistry/*metabolism, Protein Folding, Protein Structure, Tertiary/physiology, Scattering, Small Angle, X-Ray Diffraction

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Kinetic instability of the serpin Z alpha1-antitrypsin promotes aggregation.

Publication Date: 2010 Feb 19 PMID: 19944704
Authors: Knaupp, A. S. - Levina, V. - Robertson, A. L. - Pearce, M. C. - Bottomley, S. P.
Journal: J Mol Biol

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.

MeSH Categories: *Chemical Precipitation, Humans, Kinetics, Models, Molecular, Protein Conformation, Protein Folding, Protein Isoforms/chemistry/metabolism, Protein Stability, Serpins/chemistry/metabolism, Thermodynamics, alpha 1-Antitrypsin/*chemistry/*metabolism

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Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.

Publication Date: 2010 Feb 19 PMID: 19944703
Authors: Marston, J. P. - Cliff, M. J. - Reed, M. A. - Blackburn, G. M. - Hounslow, A. M. - Craven, C. J. - Waltho, J. P.
Journal: J Mol Biol

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.

MeSH Categories: Adenosine Diphosphate/chemistry/metabolism, Binding Sites, Catalysis, Crystallography, X-Ray, Geobacillus stearothermophilus/enzymology, Glyceric Acids/chemistry/metabolism, Models, Biological, Models, Molecular, Phosphoglycerate Kinase/*chemistry/*metabolism, Protein Binding, *Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary/physiology

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The bacteriophage genome undergoes a succession of intracapsid phase transitions upon DNA ejection.

Publication Date: 2010 Feb 19 PMID: 19944702
Authors: Leforestier, A. - Livolant, F.
Journal: J Mol Biol

Double-stranded DNA bacteriophage genomes are densely packaged into capsids until the ejection is triggered upon interaction of the tail with the bacterial receptor. Using cryo-electron microscopy, we describe the organization of the genome in the full capsid of T5 and show how it undergoes a series of phase transitions upon progressive ejection when the encapsidated DNA length decreases. Monodomains of hexagonally crystallized DNA segments initially form a three-dimensional lattice of defects. The structure turns liquid crystalline (two-dimensional hexagonal and then cholesteric) and finally isotropic. These structures suggest a mechanism in which defects of the full capsid would initiate the ejection and introduce the necessary fluidity to relax the constrained mosaic crystal to let the genome start flowing out of the capsid.

MeSH Categories: Bacteriophages/chemistry/*genetics/*ultrastructure, Capsid/chemistry/*metabolism, Cryoelectron Microscopy, DNA, Viral/chemistry/*metabolism, *Genome, Viral, Models, Biological, Nucleic Acid Conformation, Virus Assembly/physiology, Virus Attachment

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Identifying and quantifying orphan protein sequences in fungi.

Publication Date: 2010 Feb 19 PMID: 19944701
Authors: Ekman, D. - Elofsson, A.
Journal: J Mol Biol

For large regions of many proteins, and even entire proteins, no homology to known domains or proteins can be detected. These sequences are often referred to as orphans. Surprisingly, it has been reported that the large number of orphans is sustained in spite of a rapid increase of available genomic sequences. However, it is believed that de novo creation of coding sequences is rare in comparison to mechanisms such as domain shuffling and gene duplication; hence, most sequences should have homologs in other genomes. To investigate this, the sequences of 19 complete fungi genomes were compared. By using the phylogenetic relationship between these genomes, we could identify potentially de novo created orphans in Saccharomyces cerevisiae. We found that only a small fraction, <2%, of the S. cerevisiae proteome is orphan, which confirms that de novo creation of coding sequences is indeed rare. Furthermore, we found it necessary to compare the most closely related species to distinguish between de novo created sequences and rapidly evolving sequences where homologs are present but cannot be detected. Next, the orphan proteins (OPs) and orphan domains (ODs) were characterized. First, it was observed that both OPs and ODs are short. In addition, at least some of the OPs have been shown to be functional in experimental assays, showing that they are not pseudogenes. Furthermore, in contrast to what has been reported before and what is seen for older orphans, S. cerevisiae specific ODs and proteins are not more disordered than other proteins. This might indicate that many of the older, and earlier classified, orphans indeed are fast-evolving sequences. Finally, >90% of the detected ODs are located at the protein termini, which suggests that these orphans could have been created by mutations that have affected the start or stop codons.

MeSH Categories: Fungal Proteins/*analysis/chemistry/genetics, Fungi/genetics/*metabolism, Genome, Fungal, Open Reading Frames, Phylogeny, Protein Structure, Tertiary, Pseudogenes/genetics, Sequence Analysis, DNA, *Sequence Analysis, Protein, Sequence Homology

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Stability and DNA-binding ability of the bZIP dimers formed by the ATF-2 and c-Jun transcription factors.

Publication Date: 2010 Feb 19 PMID: 19944700
Authors: Carrillo, R. J. - Dragan, A. I. - Privalov, P. L.
Journal: J Mol Biol

The dimer formed by the ATF-2 and c-Jun transcription factors is one of the main components of the human interferon-beta enhanceosome. Although these two transcription factors are able to form two homodimers and one heterodimer, it is mainly the heterodimer that participates in the formation of this enhanceosome, binding specifically to the positive regulatory domain IV (PRDIV) site of the enhancer DNA. To understand this surprising advantage of the heterodimer, we investigated the association of these transcription factors using fragments containing the basic DNA-recognition segment and the basic leucine zipper domain (bZIP). It was found that the probability of forming the hetero-bZIP significantly exceeds the probability of forming homo-bZIPs, and that the hetero-bZIP interacts more strongly with the PRDIV site of the interferon-beta enhancer, especially in the orientation that places the folded ATF-2 basic segment in the upstream half of this asymmetric site. The effect of salt on the formation of the ATF-2/c-Jun dimer and on its ability to bind the target PRDIV site showed that electrostatic interactions between the charged groups of these proteins and with DNA play an essential role in the formation of the asymmetric ATF-2/c-Jun/PRDIV complex.

MeSH Categories: Activating Transcription Factor 2/*chemistry/*metabolism, Amino Acid Sequence, Basic-Leucine Zipper Transcription Factors/chemistry/metabolism, DNA/*metabolism, DNA-Binding Proteins/chemistry/metabolism, Humans, Protein Binding, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Multimerization/physiology, Protein Stability, Proto-Oncogene Proteins c-jun/*chemistry/*metabolism, Transcription Factors/chemistry/metabolism

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Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.

Publication Date: 2010 Feb 19 PMID: 19944699
Authors: Lewis, H. A. - Wang, C. - Zhao, X. - Hamuro, Y. - Conners, K. - Kearins, M. C. - Lu, F. - Sauder, J. M. - Molnar, K. S. - Coales, S. J. - Maloney, P. C. - Guggino, W. B. - Wetmore, D. R. - Weber, P. C. - Hunt, J. F.
Journal: J Mol Biol

The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

MeSH Categories: Crystallography, X-Ray, Cystic Fibrosis/genetics, Cystic Fibrosis Transmembrane Conductance, Regulator/*chemistry/genetics/metabolism, *Deuterium Exchange Measurement/methods, Humans, *Mass Spectrometry/methods, Models, Biological, Models, Molecular, Molecular Dynamics Simulation, Mutation/physiology, Nucleotides/*metabolism, *Protein Interaction Domains and Motifs/genetics, Protein Structure, Quaternary

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Nucleotide binding states of subunit A of the A-ATP synthase and the implication of P-loop switch in evolution.

Publication Date: 2010 Feb 19 PMID: 19944110
Authors: Kumar, A. - Manimekalai, M. S. - Balakrishna, A. M. - Jeyakanthan, J. - Gruber, G.
Journal: J Mol Biol

The crystal structures of the nucleotide-empty (A(E)), 5'-adenylyl-beta,gamma-imidodiphosphate (A(PNP))-bound, and ADP (A(DP))-bound forms of the catalytic A subunit of the energy producer A(1)A(O) ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 A and 2.4 A resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A(E) form, the phosphate analog SO(4)(2-) binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-A structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic beta subunits of F(1)F(O) ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A(1)A(O) ATP synthases, F(1)F(O) ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.

MeSH Categories: Amino Acid Sequence, Amino Acid Substitution/genetics/physiology, Binding Sites/genetics, Catalytic Domain, Crystallography, X-Ray, *Evolution, Molecular, Models, Molecular, Mutant Proteins/genetics, Nucleotides/chemistry/*metabolism, Protein Binding, Protein Structure, Secondary, Protein Subunits/chemistry/genetics/metabolism, Proton-Translocating ATPases/*chemistry/*genetics/metabolism, Pyrococcus horikoshii/enzymology/genetics, Sequence Homology, Amino Acid

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Structural basis of the action of glucosyltransferase Lgt1 from Legionella pneumophila.

Publication Date: 2010 Feb 19 PMID: 19941871
Authors: Lu, W. - Du, J. - Stahl, M. - Tzivelekidis, T. - Belyi, Y. - Gerhardt, S. - Aktories, K. - Einsle, O.
Journal: J Mol Biol

The glucosyltransferase Lgt1 is one of three glucosylating toxins of Legionella pneumophila, the causative agent of Legionnaires disease. It acts through specific glucosylation of a serine residue (S53) in the eukaryotic elongation factor 1A and belongs to type A glycosyltransferases. High-resolution crystal structures of Lgt1 show an elongated shape of the protein, with the binding site for uridine disphosphate glucose at the bottom of a deep cleft. Lgt1 shows only a low sequence identity with other type A glycosyltransferases, and structural conservation is limited to a central folding core that is usually observed within this family of proteins. Domains and protrusions added to the core motif represent determinants for the specific recognition and binding of the target. Manual docking experiments based on the crystal structures of toxin and target protein suggest an obvious mode of binding to the target that allows for efficient transfer of a glucose moiety.

MeSH Categories: Catalytic Domain, Glucosyltransferases/*chemistry/metabolism, Legionella pneumophila/*enzymology, Models, Molecular, Protein Binding, Protein Folding, Protein Structure, Secondary, Structural Homology, Protein, Uridine Diphosphate Glucose/chemistry/metabolism

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Crystal structures of an oligopeptide-binding protein from the biosynthetic pathway of the beta-lactamase inhibitor clavulanic acid.

Publication Date: 2010 Feb 19 PMID: 19941870
Authors: Mackenzie, A. K. - Valegard, K. - Iqbal, A. - Caines, M. E. - Kershaw, N. J. - Jensen, S. E. - Schofield, C. J. - Andersson, I.
Journal: J Mol Biol

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.

MeSH Categories: Arginine/chemistry/metabolism, Bacterial Proteins/*chemistry/metabolism, Carrier Proteins/*chemistry/metabolism, Catalytic Domain, Clavulanic Acid/*biosynthesis, Crystallography, X-Ray, Lipoproteins/*chemistry/metabolism, Metabolic Networks and Pathways/physiology, Models, Molecular, Protein Binding, Protein Conformation, Streptomyces/enzymology/metabolism, beta-Lactamases/*antagonists & inhibitors

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A single mutation at the sheet switch region results in conformational changes favoring lambda6 light-chain fibrillogenesis.

Publication Date: 2010 Feb 19 PMID: 19941869
Authors: Hernandez-Santoyo, A. - del Pozo Yauner, L. - Fuentes-Silva, D. - Ortiz, E. - Rudino-Pinera, E. - Sanchez-Lopez, R. - Horjales, E. - Becerril, B. - Rodriguez-Romero, A.
Journal: J Mol Biol

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda6 LCs.

MeSH Categories: Amyloid/*chemistry/*genetics/metabolism, Amyloidosis/genetics, Crystallography, X-Ray, Humans, Immunoglobulin Variable Region/chemistry/genetics/metabolism, Immunoglobulin lambda-Chains/*chemistry/*genetics/metabolism, Models, Molecular, Mutagenesis, Site-Directed, *Point Mutation/physiology, Protein Folding, Protein Multimerization/*genetics, Protein Structure, Quaternary, Protein Structure, Secondary/genetics, Protein Structure, Tertiary/genetics, Temperature, Thermodynamics

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Response acceleration in post-translationally regulated genetic circuits.

Publication Date: 2010 Mar 12 PMID: 19932119
Authors: Mitrophanov, A. Y. - Groisman, E. A.
Journal: J Mol Biol

Transcription factors must often be chemically modified to perform their functions. Yet, it is not known whether the mechanisms that bring about such modifications impact the quantitative or kinetic properties of gene expression. Phosphorylation controls the activity of regulatory proteins of the two-component system family, which constitutes a prevalent form of bacterial signal transduction. These proteins are phosphorylated/dephosphorylated by cognate sensor proteins in response to specific signals. The phosphorylation level of the regulatory proteins is also modulated by small proteins-termed connectors-that are produced when a cell experiences signals other than those detected directly by the sensors. Here, we explore how differences in the targets (i.e., sensor or regulator) and the mechanisms used by connectors to generate phosphorylated regulatory proteins affect the output of two-component systems. Our mathematical modeling demonstrates that sensor-targeting mechanisms exhibit stronger response acceleration than those where the connector targets the regulator. These differences are robust to perturbations in kinetic parameters but dependent upon the specific sensor-to-regulator ratio and how the ratio is controlled in living cells. In contrast, the steady-state output levels of the circuits are determined primarily by the circuit parameters, and can be adjusted without affecting response acceleration. Likewise, the analyzed connector-mediated circuits exhibit similar noise generation properties. Our results highlight the relationship between the architecture of genetic regulatory circuits and their dynamic properties.

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Protein-precursor tRNA contact leads to sequence-specific recognition of 5' leaders by bacterial ribonuclease P.

Publication Date: 2010 Feb 12 PMID: 19932118
Authors: Koutmou, K. S. - Zahler, N. H. - Kurz, J. C. - Campbell, F. E. - Harris, M. E. - Fierke, C. A.
Journal: J Mol Biol

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.

MeSH Categories: 5' Untranslated Regions/*genetics, Adenosine/metabolism, Amino Acid Substitution/drug effects, Bacillus subtilis/*enzymology/genetics, Base Sequence, Calcium/pharmacology, Escherichia coli/*enzymology/genetics, Genome, Bacterial, Molecular Sequence Data, Mutant Proteins/chemistry/metabolism, Nucleotides/metabolism, Protein Binding/drug effects, Protein Structure, Secondary, RNA Precursors/*metabolism, RNA, Transfer/genetics, Ribonuclease P/chemistry/*metabolism, Substrate Specificity/drug effects

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Cation-selective pathway of OmpF porin revealed by anomalous X-ray diffraction.

Publication Date: 2010 Feb 19 PMID: 19932117
Authors: Dhakshnamoorthy, B. - Raychaudhury, S. - Blachowicz, L. - Roux, B.
Journal: J Mol Biol

The OmpF porin from the Escherichia coli outer membrane folds into a trimer of beta-barrels, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the beta-barrel pore to form a narrow "constriction zone". A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the beta-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P6(3) space group and two different data sets were obtained at 2.6 A and 3.0 A resolution with K(+) and Rb(+), respectively. The Rb(+)-soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 A and cation positions were determined. A PEG molecule was observed in the pore region for both the K(+) and Rb(+)-soaked crystals, where it interacts with loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.

MeSH Categories: Cations/*metabolism/pharmacology, Chlorides/chemistry/metabolism, Crystallography, X-Ray, Models, Molecular, Polyethylene Glycols/chemistry/metabolism/pharmacology, Porins/*chemistry/metabolism, Potassium Chloride/chemistry/metabolism, Protein Structure, Quaternary/drug effects, Rubidium/chemistry/metabolism, Signal Transduction, Substrate Specificity, X-Ray Diffraction/*methods

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Structural mechanics of DNA wrapping in the nucleosome.

Publication Date: 2010 Feb 19 PMID: 19932116
Authors: Battistini, F. - Hunter, C. A. - Gardiner, E. J. - Packer, M. J.
Journal: J Mol Biol

Experimental X-ray crystal structures and a database of calculated structural parameters of DNA octamers were used in combination to analyse the mechanics of DNA bending in the nucleosome core complex. The 1kx5 X-ray crystal structure of the nucleosome core complex was used to determine the relationship between local structure at the base-step level and the global superhelical conformation observed for nucleosome-bound DNA. The superhelix is characterised by a large curvature (597 degrees) in one plane and very little curvature (10 degrees) in the orthogonal plane. Analysis of the curvature at the level of 10-step segments shows that there is a uniform curvature of 30 degrees per helical turn throughout most of the structure but that there are two sharper kinks of 50 degrees at +/-2 helical turns from the central dyad base pair. The curvature is due almost entirely to the base-step parameter roll. There are large periodic variations in roll, which are in phase with the helical twist and account for 500 degrees of the total curvature. Although variations in the other base-step parameters perturb the local path of the DNA, they make minimal contributions to the total curvature. This implies that DNA bending in the nucleosome is achieved using the roll-slide-twist degree of freedom previously identified as the major degree of freedom in naked DNA oligomers. The energetics of bending into a nucleosome-bound conformation were therefore analysed using a database of structural parameters that we have previously developed for naked DNA oligomers. The minimum energy roll, the roll flexibility force constant and the maximum and minimum accessible roll values were obtained for each base step in the relevant octanucleotide context to account for the effects of conformational coupling that vary with sequence context. The distribution of base-step roll values and corresponding strain energy required to bend DNA into the nucleosome-bound conformation defined by the 1kx5 structure were obtained by applying a constant bending moment. When a single bending moment was applied to the entire sequence, the local details of the calculated structure did not match the experiment. However, when local 10-step bending moments were applied separately, the calculated structure showed excellent agreement with experiment. This implies that the protein applies variable bending forces along the DNA to maintain the superhelical path required for nucleosome wrapping. In particular, the 50 degrees kinks are constraints imposed by the protein rather than a feature of the 1kx5 DNA sequence. The kinks coincide with a relatively flexible region of the sequence, and this is probably a prerequisite for high-affinity nucleosome binding, but the bending strain energy is significantly higher at these points than for the rest of the sequence. In the most rigid regions of the sequence, a higher strain energy is also required to achieve the standard 30 degrees curvature per helical turn. We conclude that matching of the DNA sequence to the local roll periodicity required to achieve bending, together with the increased flexibility required at the kinks, determines the sequence selectivity of DNA wrapping in the nucleosome.

MeSH Categories: Biomechanics, Chromatin Assembly and Disassembly/physiology, Crystallography, X-Ray, DNA/*chemistry/*metabolism, Models, Biological, Models, Molecular, Models, Theoretical, Molecular Dynamics Simulation, *Nucleic Acid Conformation, Nucleosomes/chemistry/*metabolism, Protein Structure, Quaternary

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The effect of hydrophilic substitutions and anionic lipids upon the transverse positioning of the transmembrane helix of the ErbB2 (neu) protein incorporated into model membrane vesicles.

Publication Date: 2010 Feb 12 PMID: 19931543
Authors: Shahidullah, K. - Krishnakumar, S. S. - London, E.
Journal: J Mol Biol

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H>E>Q>K>G>V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.

MeSH Categories: Amino Acid Sequence, *Amino Acid Substitution/genetics, Anions, Hydrogen-Ion Concentration, Membrane Lipids/*chemistry/*metabolism, *Models, Biological, Models, Molecular, Molecular Sequence Data, Mutant Proteins/chemistry, Mutation/genetics, Peptides/chemistry, Phosphatidylcholines/chemistry, Protein Structure, Secondary, Receptor, erbB-2/*chemistry/genetics/*metabolism, Spectrometry, Fluorescence, Tryptophan/metabolism, Unilamellar Liposomes/*chemistry

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Dimerisation of the UBA domain of p62 inhibits ubiquitin binding and regulates NF-kappaB signalling.

Publication Date: 2010 Feb 12 PMID: 19931284
Authors: Long, J. - Garner, T. P. - Pandya, M. J. - Craven, C. J. - Chen, P. - Shaw, B. - Williamson, M. P. - Layfield, R. - Searle, M. S.
Journal: J Mol Biol

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappaB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappaB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K(dim) approximately 4-12 microM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappaB luciferase reporter assays, are associated with reduced NF-kappaB activity compared with wt-p62.

MeSH Categories: Adaptor Proteins, Signal Transducing/*chemistry/*metabolism, Biophysical Phenomena, Cell Line, Humans, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Mutation/genetics, NF-kappa B/*metabolism, Polyubiquitin/metabolism, Protein Binding, *Protein Multimerization, Protein Stability, Protein Structure, Secondary, Protein Structure, Tertiary, *Signal Transduction, Solutions, Ubiquitin/*metabolism

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An RNA structural switch regulates diploid genome packaging by Moloney murine leukemia virus.

Publication Date: 2010 Feb 12 PMID: 19931283
Authors: Miyazaki, Y. - Garcia, E. L. - King, S. R. - Iyalla, K. - Loeliger, K. - Starck, P. - Syed, S. - Telesnitsky, A. - Summers, M. F.
Journal: J Mol Biol

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.

MeSH Categories: Base Sequence, Binding Sites, Cell Line, Dimerization, *Diploidy, Genome, Viral/*genetics, Humans, Molecular Sequence Data, Moloney murine leukemia virus/*genetics/*physiology, Mutation/genetics, Nucleic Acid Conformation, Nucleocapsid/genetics, RNA Stability, RNA, Viral/*chemistry/*genetics, Temperature, Transcription, Genetic, Virus Assembly/*genetics

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Structure and dynamics of the actin filament.

Publication Date: 2010 Feb 19 PMID: 19931282
Authors: Pfaendtner, J. - Lyman, E. - Pollard, T. D. - Voth, G. A.
Journal: J Mol Biol

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of gamma-phosphate from ADP-P(i) actin.

MeSH Categories: Actins/chemistry/metabolism, Animals, Computer Simulation, Crystallography, X-Ray, Eukaryotic Cells/metabolism, Humans, Microfilaments/*chemistry/*metabolism, Models, Molecular, Molecular Dynamics Simulation, Nucleotides/metabolism, Phalloidine/chemistry/metabolism, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs/physiology, Protein Multimerization/physiology, Structure-Activity Relationship

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Membrane insertion of marginally hydrophobic transmembrane helices depends on sequence context.

Publication Date: 2010 Feb 12 PMID: 19931281
Authors: Hedin, L. E. - Ojemalm, K. - Bernsel, A. - Hennerdal, A. - Illergard, K. - Enquist, K. - Kauko, A. - Cristobal, S. - von Heijne, G. - Lerch-Bader, M. - Nilsson, I. - Elofsson, A.
Journal: J Mol Biol

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

MeSH Categories: Amino Acid Sequence, Animals, Biological Assay, Cell Membrane/*metabolism, Humans, *Hydrophobicity, Protein Structure, Secondary, Proteins/*chemistry/*metabolism, Thermodynamics

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Stochastic switching in gene networks can occur by a single-molecule event or many molecular steps.

Publication Date: 2010 Feb 12 PMID: 19931280
Authors: Choi, P. J. - Xie, X. S. - Shakhnovich, E. I.
Journal: J Mol Biol

Due to regulatory feedback, biological networks can exist stably in multiple states, leading to heterogeneous phenotypes among genetically identical cells. Random fluctuations in protein numbers, tuned by specific molecular mechanisms, have been hypothesized to drive transitions between these different states. We develop a minimal theoretical framework to analyze the limits of switching in terms of simple experimental parameters. Our model identifies and distinguishes between two distinct molecular mechanisms for generating stochastic switches. In one class of switches, the stochasticity of a single-molecule event, a specific and rare molecular reaction, directly controls the macroscopic change in a cell's state. In the second class, no individual molecular event is significant, and stochasticity arises from the propagation of biochemical noise through many molecular pathways and steps. As an example, we explore switches based on protein-DNA binding fluctuations and predict relations between transcription factor kinetics, absolute switching rate, robustness, and efficiency that differentiate between switching by single-molecule events or many molecular steps. Finally, we apply our methods to recent experimental data on switching in Escherichia coli lactose metabolism, providing quantitative interpretations of a single-molecule switching mechanism.

MeSH Categories: Escherichia coli/*genetics, Feedback, Physiological, Gene Expression Regulation, Bacterial, Gene Regulatory Networks/*genetics, Kinetics, Lac Operon/genetics, Models, Genetic, Protein Biosynthesis, Stochastic Processes, Transcription Factors/metabolism, Transcription, Genetic

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Organization, structure, and assembly of alpha-carboxysomes determined by electron cryotomography of intact cells.

Publication Date: 2010 Feb 12 PMID: 19925807
Authors: Iancu, C. V. - Morris, D. M. - Dou, Z. - Heinhorst, S. - Cannon, G. C. - Jensen, G. J.
Journal: J Mol Biol

Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby carboxysomes. Small granular bodies were also seen within carboxysomes. These observations suggest a functional relationship between carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial carboxysome was ever observed per cell. Based on these observations, we propose a model for carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.

MeSH Categories: Bacteria/*cytology/*ultrastructure, *Cryoelectron Microscopy, Cytoplasmic Granules/metabolism/ultrastructure, Elements, Inclusion Bodies/*ultrastructure, Polyphosphates/metabolism, *Tomography

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The ligand-free state of the TPP riboswitch: a partially folded RNA structure.

Publication Date: 2010 Feb 12 PMID: 19925806
Authors: Ali, M. - Lipfert, J. - Seifert, S. - Herschlag, D. - Doniach, S.
Journal: J Mol Biol

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.

MeSH Categories: Aptamers, Nucleotide/chemistry, Arabidopsis/genetics, Base Sequence, Ligands, Magnesium/pharmacology, Models, Molecular, Molecular Sequence Data, *Nucleic Acid Conformation, RNA/*chemistry, Scattering, Small Angle, Solutions, Thiamine Pyrophosphate/*chemistry, X-Ray Diffraction

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E6 proteins from diverse papillomaviruses self-associate both in vitro and in vivo.

Publication Date: 2010 Feb 12 PMID: 19917295
Authors: Zanier, K. - Ruhlmann, C. - Melin, F. - Masson, M. - Ould M'hamed Ould Sidi, A. - Bernard, X. - Fischer, B. - Brino, L. - Ristriani, T. - Rybin, V. - Baltzinger, M. - Vande Pol, S. - Hellwig, P. - Schultz, P. - Trave, G.
Journal: J Mol Biol

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.

MeSH Categories: Carrier Proteins/metabolism, Cell Nucleus/metabolism, Chromatography, Gel, Humans, Mutant Proteins/chemistry/metabolism, Protein Binding, Protein Stability, Protein Structure, Quaternary, Protein Structure, Secondary, Recombinant Fusion Proteins/chemistry/metabolism, Solubility, Spectroscopy, Fourier Transform Infrared, Viral Proteins/chemistry/*metabolism/ultrastructure, Zinc/chemistry

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The unique kinetics of iron release from transferrin: the role of receptor, lobe-lobe interactions, and salt at endosomal pH.

Publication Date: 2010 Feb 12 PMID: 19917294
Authors: Byrne, S. L. - Chasteen, N. D. - Steere, A. N. - Mason, A. B.
Journal: J Mol Biol

Transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor-mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions, and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or diferric hTF (with iron locked in one lobe) provided distinct kinetic end points, allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped-flow spectrofluorimetry, in the presence and in the absence of the TFR, unequivocally establish the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time, the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe(2)hTF. Calculated cooperativity factors, in addition to plots of time-dependent species distributions in the absence and in the presence of the TFR, clearly illustrate the differences. Accurate rate constants for the pH and salt-induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.

MeSH Categories: Endosomes/*drug effects/*metabolism, Humans, Hydrogen-Ion Concentration/drug effects, Kinetics, Potassium Chloride/*pharmacology, Protein Binding/drug effects, Protein Structure, Secondary, Receptors, Transferrin/*chemistry/*metabolism, Solubility/drug effects, Time Factors, Transferrin/*metabolism

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Ribosomal interaction of Bacillus stearothermophilus translation initiation factor IF2: characterization of the active sites.

Publication Date: 2010 Feb 12 PMID: 19917289
Authors: Caserta, E. - Ferrara, C. - Milon, P. - Fabbretti, A. - Rocchetti, A. - Tomsic, J. - Pon, C. L. - Gualerzi, C. O. - La Teana, A.
Journal: J Mol Biol

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive "cross-talking" between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.

MeSH Categories: Amino Acid Substitution/genetics, *Catalytic Domain, Dipeptides/metabolism, Escherichia coli/growth & development, Genes, Bacterial, Genetic Complementation Test, Geobacillus stearothermophilus/genetics/*metabolism, Kinetics, Mutant Proteins/chemistry/metabolism, Prokaryotic Initiation Factor-2/*chemistry/genetics/*metabolism, Protein Binding, Protein Biosynthesis, Protein Structure, Secondary, Ribosome Subunits/metabolism, Ribosomes/*metabolism, Sequence Deletion

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Residue-specific analysis of frustration in the folding landscape of repeat beta/alpha protein apoflavodoxin.

Publication Date: 2010 Feb 12 PMID: 19913555
Authors: Stagg, L. - Samiotakis, A. - Homouz, D. - Cheung, M. S. - Wittung-Stafshede, P.
Journal: J Mol Biol

Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding.

MeSH Categories: Amino Acids/*chemistry, Apoproteins/*chemistry/genetics, Circular Dichroism, Computational Biology, Desulfovibrio desulfuricans/*chemistry, Flavodoxin/*chemistry/genetics, Kinetics, Mutant Proteins/chemistry/genetics, Mutation/genetics, *Protein Folding, Protein Stability, Protein Structure, Secondary, *Repetitive Sequences, Amino Acid, Temperature

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Bacterial pleckstrin homology domains: a prokaryotic origin for the PH domain.

Publication Date: 2010 Feb 12 PMID: 19913036
Authors: Xu, Q. - Bateman, A. - Finn, R. D. - Abdubek, P. - Astakhova, T. - Axelrod, H. L. - Bakolitsa, C. - Carlton, D. - Chen, C. - Chiu, H. J. - Chiu, M. - Clayton, T. - Das, D. - Deller, M. C. - Duan, L. - Ellrott, K. - Ernst, D. - Farr, C. L. - Feuerhelm, J. - Grant, J. C. - Grzechnik, A. - Han, G. W. - Jaroszewski, L. - Jin, K. K. - Klock, H. E. - Knuth, M. W. - Kozbial, P. - Krishna, S. S. - Kumar, A. - Marciano, D. - McMullan, D. - Miller, M. D. - Morse, A. T. - Nigoghossian, E. - Nopakun, A. - Okach, L. - Puckett, C. - Reyes, R. - Rife, C. L. - Sefcovic, N. - Tien, H. J. - Trame, C. B. - van den Bedem, H. - Weekes, D. - Wooten, T. - Hodgson, K. O. - Wooley, J. - Elsliger, M. A. - Deacon, A. M. - Godzik, A. - Lesley, S. A. - Wilson, I. A.
Journal: J Mol Biol

Pleckstrin homology (PH) domains have been identified only in eukaryotic proteins to date. We have determined crystal structures for three members of an uncharacterized protein family (Pfam PF08000), which provide compelling evidence for the existence of PH-like domains in bacteria (PHb). The first two structures contain a single PHb domain that forms a dome-shaped, oligomeric ring with C(5) symmetry. The third structure has an additional helical hairpin attached at the C-terminus and forms a similar but much larger ring with C(12) symmetry. Thus, both molecular assemblies exhibit rare, higher-order, cyclic symmetry but preserve a similar arrangement of their PHb domains, which gives rise to a conserved hydrophilic surface at the intersection of the beta-strands of adjacent protomers that likely mediates protein-protein interactions. As a result of these structures, additional families of PHb domains were identified, suggesting that PH domains are much more widespread than originally anticipated. Thus, rather than being a eukaryotic innovation, the PH domain superfamily appears to have existed before prokaryotes and eukaryotes diverged.

MeSH Categories: Amino Acid Sequence, Bacteria/*metabolism, Bacterial Proteins/*chemistry, Binding Sites, Conserved Sequence, Crystallography, X-Ray, Eukaryotic Cells, *Evolution, Molecular, Models, Molecular, Molecular Sequence Data, Prokaryotic Cells/*metabolism, Protein Binding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, *Sequence Homology, Amino Acid, Surface Properties

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Structural bases for stability-function tradeoffs in antibiotic resistance.

Publication Date: 2010 Feb 12 PMID: 19913034
Authors: Thomas, V. L. - McReynolds, A. C. - Shoichet, B. K.
Journal: J Mol Biol

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization "strain" of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in beta-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of beta-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C beta-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol(-)(1) to 4.1 kcal mol(-)(1)), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 A from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.

MeSH Categories: Amino Acid Substitution, Anti-Bacterial Agents/chemistry, Apoenzymes/chemistry, Cefotaxime/chemistry, Crystallography, X-Ray, *Drug Resistance, Microbial, Enzyme Stability, Kinetics, Models, Molecular, Mutant Proteins/chemistry, Pliability, Protein Structure, Secondary, Substrate Specificity, Thermodynamics, beta-Lactamases/*chemistry/*metabolism

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Lability landscape and protease resistance of human insulin amyloid: a new insight into its molecular properties.

Publication Date: 2010 Feb 12 PMID: 19913026
Authors: Malisauskas, M. - Weise, C. - Yanamandra, K. - Wolf-Watz, M. - Morozova-Roche, L.
Journal: J Mol Biol

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.

MeSH Categories: Amyloid/*chemistry/*metabolism/ultrastructure, Circular Dichroism, Electrophoresis, Endopeptidase K/*metabolism, Enzyme Stability, Humans, Hydrogen-Ion Concentration, Insulin/*chemistry/*metabolism, Kinetics, Microscopy, Atomic Force, *Models, Molecular, Protein Binding, Protein Structure, Quaternary, Solutions, Spectrum Analysis, Thiazoles/metabolism, Time Factors

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Compensated pathogenic deviations: analysis of structural effects.

Publication Date: 2010 Feb 12 PMID: 19900462
Authors: Baresic, A. - Hopcroft, L. E. - Rogers, H. H. - Hurst, J. M. - Martin, A. C.
Journal: J Mol Biol

Pathogenic deviations (PDs) in humans are disease-causing missense mutations. However, in some cases, these disease-associated residues occur as the wild-type residues in functionally equivalent proteins in other species and these cases are termed 'compensated pathogenic deviations' (CPDs). The lack of pathogenicity in a non-human protein is presumed to be explained in most cases by the presence of compensatory mutations, most commonly within the same protein. Identification of structural features of CPDs and detection of specific compensatory events will help us to understand traversal along fitness landscape valleys in protein evolution. We divided mutations listed in the OMIM (Online Mendelian Inheritance in Man) database into PD and CPD data sets and performed two independent analyses: (i) We searched for potential compensatory mutations spatially close to the CPDs and, (ii) using our SAAPdb database, we examined likely structural effects to try to explain why mutations are pathogenic, comparing PDs and CPDs. Our data sets were obtained from a set of 245 human proteins of known structure and contained a total of 2328 mutations of which 453 (from 85 structures) were seen to be compensated in at least one functionally equivalent protein in another (non-human) species. Structural analysis results confirm previous findings that CPDs are, on average, 'milder' in their likely structural effects than uncompensated PDs and tend to be on the protein surface. We also showed that the residues surrounding the CPD residue in the folded protein are more often mutated than the residues surrounding an uncompensated mutation, supporting the hypothesis that compensation is largely a result of structurally local mutations.

MeSH Categories: Amino Acid Sequence, Animals, Antithrombin III/chemistry/genetics, Binding Sites, Databases, Protein, Disease/*genetics, Humans, Models, Molecular, Molecular Sequence Data, Mutation, Missense/*genetics, Protein Binding, Protein Folding, Protein Stability, Reproducibility of Results, Sheep

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Triosephosphate Isomerase: (15)N and (13)C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy.

Publication Date: 2010 Mar 19 PMID: 19854202
Authors: Xu, Y. - Lorieau, J. - McDermott, A. E.
Journal: J Mol Biol

Microcrystalline uniformly (13)C,(15)N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.

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