Journal of Molecular Biology

Structures and Free-Energy Landscapes of the Wild Type and Mutants of the Abeta(21-30) Peptide Are Determined by an Interplay between Intrapeptide Electrostatic and Hydrophobic Interactions.

Publication Date: 2008 May 12 PMID: 18479708
Authors: Tarus, B. - Straub, J. E. - Thirumalai, D.
Journal: J Mol Biol

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Abeta(21-30) peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK(a) values that compare well with the experimental estimates. The pK(a) values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in "dried" salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N
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Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial FAS II Pathway: New Insights into Its Substrate Recognition Properties.

Publication Date: 2008 Apr 24 PMID: 18479707
Authors: Chen, Z. J. - Pudas, R. - Sharma, S. - Smart, O. S. - Juffer, A. H. - Hiltunen, J. K. - Wierenga, R. K. - Haapalainen, A. M.
Journal: J Mol Biol

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.

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Molecular Dynamics-Solvated Interaction Energy Studies of Protein-Protein Interactions: The MP1-p14 Scaffolding Complex.

Publication Date: 2008 Apr 20 PMID: 18479705
Authors: Cui, Q. - Sulea, T. - Schrag, J. D. - Munger, C. - Hung, M. N. - Naim, M. - Cygler, M. - Purisima, E. O.
Journal: J Mol Biol

Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energy functions and two different single-structure representations of the complex. Refined binding affinity predictions for select hot-spot mutations are carried out by applying first-principle methods such as the molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) to the molecular dynamics (MD) trajectories for mutated and wild-type complexes. Here, predicted hot-spot residues were actually mutated to alanine, and crystal structures of the mutated complexes were determined. Two mutated MP1-p14 complexes were investigated, the p14(Y56A)-mutated complex and the MP1(L63A,L65A)-mutated complex. Alternative ways to generate MD ensembles for mutant complexes, not relying on crystal structures for mutated complexes, were also investigated. The SIE function, fitted on protein-ligand binding affinities, gave absolute binding affinity predictions in excellent agreement with experiment and outperformed standard MM-GBSA predictions when tested on the MD ensembles of Ras-Raf and Ras-RalGDS protein-protein complexes. For wild-type and mutant MP1-p14 complexes, SIE predictions of relative binding affinities were supported by a yeast two-hybrid assay that provided semiquantitative relative interaction strengths. Results on the MP1-mutated complex suggested that SIE predictions deteriorate if mutant MD ensembles are approximated by just mutating the wild-type MD trajectory. The SIE data on the p14-mutated complex indicated feasibility for generating mutant MD ensembles from mutated wild-type crystal structure, despite local structural differences observed upon mutation. For energetic considerations, this would circumvent costly needs to produce and crystallize mutated complexes. The sensitized protein-protein interface afforded by the p14(Y56A) mutation identified here has practical applications in screening-based discovery of first-generation small-molecule hits for further development into specific modulators of the mitogen-activated protein kinase signaling pathway.

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The Structural Basis for the Activation and Peptide Recognition of Bacterial ClpP.

Publication Date: 2008 Apr 20 PMID: 18468623
Authors: Kim, D. Y. - Kim, K. K.
Journal: J Mol Biol

ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP.

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Structural and Biochemical Characterization of a Novel Aldehyde Dehydrogenase Encoded by the Benzoate Oxidation Pathway in Burkholderia xenovorans LB400.

Publication Date: 2008 Apr 18 PMID: 18462753
Authors: Bains, J. - Boulanger, M. J.
Journal: J Mol Biol

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH(C)) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD(+) and NADP(+)). Our data definitively show that ALDH(C) is more active in the presence of NADP(+) and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-A crystal structure of ALDH(C) in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2'-phosphoryl moiety of NADP(+) and a threonine/lysine pair on ALDH(C). The catalytic preference of ALDH(C) for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH(C) induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH(C) in benzoate degradation.

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Allosteric and Electrostatic Protein-Protein Interactions Regulate the Assembly of the Heterohexameric Tim9-Tim10 Complex.

Publication Date: 2008 May 5 PMID: 18462749
Authors: Ivanova, E. - Lu, H.
Journal: J Mol Biol

Protein-protein interactions are crucial processes in virtually all cellular events. The heterohexameric Tim9-Tim10 complex of the mitochondrial intermembrane space plays an important role during import of mitochondrial membrane proteins. It consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While the individual protein Tim9 forms a homodimer, Tim10 is a monomer. Further to our previous investigation on the complex formation pathway, in this study, the assembly mechanism of Tim9-Tim10 was investigated using a stopped-flow technique coupled with mutagenesis. We show that while the initial velocity of the assembly depends on Tim9 concentration linearly, it presents a sigmoid curve on Tim10. In addition, the overall rate of assembly depends on the pH level in a bell-shaped profile, and two pK(a) values that are in good agreement with the respective isoelectric points of Tim9 and Tim10 were determined. Using a Tim10F70W mutant, we were able to show that there was clear salt concentration dependence in the rate of assembly at the early stages. Taken together, the results of pH and salt concentration dependence indicate that electrostatic interactions are important and provide an initial driving force for the complex formation. Thus, this study not only demonstrates that allosteric and electrostatic interactions are two key regulators for the assembly of the Tim9-Tim10 complex but also has important implications for our understanding of how proteins interact with their partners.

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An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

Publication Date: 2008 May 30 PMID: 18455737
Authors: Lipes, B. D. - Chen, Y. H. - Ma, H. - Staats, H. F. - Kenan, D. J. - Gunn, M. D.
Journal: J Mol Biol

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

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Enhancement of transactivation activity of Rta of Epstein-Barr virus by RanBPM.

Publication Date: 2008 May 30 PMID: 18455188
Authors: Chang, L. K. - Liu, S. T. - Kuo, C. W. - Wang, W. H. - Chuang, J. Y. - Bianchi, E. - Hong, Y. R.
Journal: J Mol Biol

Epstein-Barr virus (EBV) expresses the immediate-early protein Rta to activate the transcription of EBV lytic genes and the lytic cycle. We show that RanBPM acts as a binding partner of Rta in yeast two-hybrid analysis. The binding was confirmed by glutathione-S-transferase pull-down assay. A coimmunoprecipitation experiment and confocal microscopy revealed that RanBPM and Rta interact in vivo and colocalize in the nucleus. The interaction appears to involve the SPRY domain in RanBPM and the region between amino acid residues 416 to 476 in Rta. The interaction promotes the transactivation activity of Rta in activating the transcription of BMLF1 and p21 in transient transfection assays. Additionally, RanBPM interacts with SUMO-E2 (Ubc9) to promote sumoylation of Rta by SUMO-1. This fact explains why the expression of RanBPM enhances the transactivation activity of Rta. Taken together, the present results indicate a new role of RanBPM in regulating a viral protein that is critical to EBV lytic activation.

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Polar, peritrichous, and lateral flagella belong to three distinguishable flagellar families.

Publication Date: 2008 May 30 PMID: 18455187
Authors: Fujii, M. - Shibata, S. - Aizawa, S.
Journal: J Mol Biol

The bacterial flagellum transforms its shape into several distinguishable helical shapes (polymorphs) under various environmental conditions. Polymorphs of each type of flagellum stay on a circle in the pitch-diameter (P versus piD) plot, indicating that they all belong to one family. Previously, we showed that the flagellar family of a marine bacterium Idiomarina loihiensis (Family II) differed from the conventional flagellar family of Salmonella typhimurium (Family I). The pitch and diameter of Family II flagella are half those of Family I flagella. We have suggested that Family I encompasses peritrichous flagella, while Family II forms a polar flagellum. In this study, we have surveyed the polymorphs of flagella from 18 other species and categorized their family types. Previous observations were confirmed; Family I form peritrichous flagella and Family II form polar flagella. Furthermore, we found that lateral flagella had helical parameters much smaller than those of the other two Families and thus belong to a new family (Family III).

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Structure and function of the arginine repressor-operator complex from Bacillus subtilis.

Publication Date: 2008 May 30 PMID: 18455186
Authors: Garnett, J. A. - Marincs, F. - Baumberg, S. - Stockley, P. G. - Phillips, S. E.
Journal: J Mol Biol

In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, the arginine repressor. In Bacillus subtilis this transcription factor is called AhrC and has roles in both the repression and activation of the genes involved in arginine metabolism. It interacts with 18 bp ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a hexamer and each subunit has two domains. The C-terminal domains form the core, mediating inter-subunit interactions and L-arginine binding, while the N-terminal domains contain a winged helix-turn-helix DNA-binding motif and are arranged around the periphery. Upon binding of the co-repressor L-arginine there is a approximately 15 degrees relative rotation between core C-terminal trimers. Here, we report the X-ray crystal structure of a dimer of the N-terminal domains of AhrC (NAhrC) in complex with an 18 bp DNA ARG box operator, refined to 2.85 A resolution. Comparison of the N-terminal domains within this complex with those of the free domain reveals that the flexible beta-wings of the DNA-binding motif in the free domain form a stable dimer interface in the protein-DNA complex, favouring correct orientation of the recognition helices. These are then positioned to insert into adjacent turns of the major groove of the ARG box, whilst the wings contact the minor groove. There are extensive contacts between the protein and the DNA phosphodiester backbone, as well as a number of direct hydrogen bonds between conserved amino acid side chains and bases. Combining this structure with other crystal structures of other AhrC components, we have constructed a model of the repression complex of AhrC at the B. subtilis biosynthetic argC operator and, along with transcriptome data, analysed the origins of sequence specificity and arginine activation.

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Crystal structure of a new type of NADPH-dependent quinone oxidoreductase (QOR2) from Escherichia coli.

Publication Date: 2008 May 30 PMID: 18455185
Authors: Kim, I. K. - Yim, H. S. - Kim, M. K. - Kim, D. W. - Kim, Y. M. - Cha, S. S. - Kang, S. O.
Journal: J Mol Biol

Escherichia coli QOR2 [NAD(P)H-dependent quinone oxidoreductase; a ytfG gene product], which catalyzes two-electron reduction of methyl-1,4-benzoquinone, is a new type of quinone-reducing enzyme with distinct primary sequence and oligomeric conformation from previously known quinone oxidoreductases. The crystal structures of native QOR2 and the QOR2-NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) complex reveal that QOR2 consists of two domains (N-domain and C-domain) resembling those of NmrA, a negative transcriptional regulator that belongs to the short-chain dehydrogenase/reductase family. The N-domain, which adopts the Rossmann fold, provides a platform for NADPH binding, whereas the C-domain, which contains a hydrophobic pocket connected to the NADPH-binding site, appears to play important roles in substrate binding. Asn143 near the NADPH-binding site has been identified to be involved in substrate binding and catalysis from structural and mutational analyses. Moreover, compared with wild-type strain, the qor2-overexpressing strain shows growth retardation and remarkable decrease in several enzymes involved in carbon metabolism, suggesting that QOR2 could play some physiological roles in addition to quinone reduction.

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Escherichia coli RNase P RNA: substrate ribose modifications at G+1, but not nucleotide -1/+73 base pairing, affect the transition state for cleavage chemistry.

Publication Date: 2008 May 23 PMID: 18452950
Authors: Cuzic, S. - Heidemann, K. A. - Wohnert, J. - Hartmann, R. K.
Journal: J Mol Biol

The temperature dependence of processing of precursor tRNA(Gly) (ptRNA(Gly)) variants carrying a single 2'-OCH(3) or locked nucleic acid (LNA) modification at G+1 by Escherichia coli endoribonuclease P RNA was studied at rate-limiting chemistry. We show, for the first time, that these ribose modifications at nucleotide +1 increase the activation energy and alter the activation parameters for the transition state of hydrolysis at the canonical (c(0)) cleavage site (between nucleotides -1 and +1). The modified substrates, particularly the one with LNA at G+1, caused an increase in the activation enthalpy Delta H(double dagger), which was partly compensated for by a simultaneous increase in the activation entropy DeltaS(double dagger). NMR imino proton spectra of model acceptor stems derived from the same ptRNA variants unveiled that a riboT or U at -1 forms two hydrogen bonds with U+73, thus extending the acceptor stem by 1 bp. The non-canonical base pair is substantially stabilized by LNA substitution at nucleotides -1 or +1. To address if the activation energy increase owing to LNA at G+1 stems from dissociation of the U(-1)-U(+73) base pair as a prerequisite for interaction of U(+73) with U294 in endoribonuclease P RNA, we tested a ptRNA(Gly) variant that is capable of forming an extra C(-1)-G(+73) Watson-Crick base pair. However, compared with a control ptRNA (C at -1, U at +73), no significant change in activation parameters was observed for this ptRNA. Thus, our results argue against the possibility that breaking of an additional base pair at the end of the acceptor stem may present an energetic barrier for reaching the transition state of the chemical step for cleavage at the canonical (c(0)) phosphodiester.

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Prediction of interacting single-stranded RNA bases by protein-binding patterns.

Publication Date: 2008 May 30 PMID: 18452949
Authors: Shulman-Peleg, A. - Shatsky, M. - Nussinov, R. - Wolfson, H. J.
Journal: J Mol Biol

Prediction of protein-RNA interactions at the atomic level of detail is crucial for our ability to understand and interfere with processes such as gene expression and regulation. Here, we investigate protein binding pockets that accommodate extruded nucleotides not involved in RNA base pairing. We observed that most of the protein-interacting nucleotides are part of a consecutive fragment of at least two nucleotides whose rings have significant interactions with the protein. Many of these share the same protein binding cavity and more than 30% of such pairs are pi-stacked. Since these local geometries cannot be inferred from the nucleotide identities, we present a novel framework for their prediction from the properties of protein binding sites. First, we present a classification of known RNA nucleotide and dinucleotide protein binding sites and identify the common types of shared 3-D physicochemical binding patterns. These are recognized by a new classification methodology that is based on spatial multiple alignment. The shared patterns reveal novel similarities between dinucleotide binding sites of proteins with different overall sequences, folds and functions. Given a protein structure, we use these patterns for the prediction of its RNA dinucleotide binding sites. Based on the binding modes of these nucleotides, we further predict an RNA fragment that interacts with those protein binding sites. With these knowledge-based predictions, we construct an RNA fragment that can have a previously unknown sequence and structure. In addition, we provide a drug design application in which the database of all known small-molecule binding sites is searched for regions similar to nucleotide and dinucleotide binding patterns, suggesting new fragments and scaffolds that can target them.

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Structural analysis of the Saf pilus by electron microscopy and image processing.

Publication Date: 2008 May 23 PMID: 18448124
Authors: Salih, O. - Remaut, H. - Waksman, G. - Orlova, E. V.
Journal: J Mol Biol

Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a "beads on a string"-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.

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Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly.

Publication Date: 2008 May 30 PMID: 18448123
Authors: Lorenzen, K. - Olia, A. S. - Uetrecht, C. - Cingolani, G. - Heck, A. J.
Journal: J Mol Biol

Large oligomeric portal assemblies have a central role in the life-cycles of bacteriophages and herpesviruses. The stoichiometry of in vitro assembled portal proteins has been a subject of debate for several years. The intrinsic polymorphic oligomerization of ectopically expressed portal proteins makes it possible to form rings of diverse stoichiometry (e.g., 11-mer, 12-mer, 13-mer, etc.) in solution. In this study, we have investigated the stoichiometry of the in vitro-assembled portal protein of bacteriophage P22 and characterized its association with the tail factor gp4. Using native mass spectrometry, we show for the first time that the reconstituted portal protein (assembled in vitro using a modified purification and assembly protocol) is exclusively dodecameric. Under the conditions used here, 12 copies of tail factor gp4 bind to the portal ring, in a cooperative fashion, to form a 12:12 complex of 1.050 MDa. We applied tandem mass spectrometry to the complete assembly and found an unusual dimeric dissociation pattern of gp4, suggesting a dimeric sub-organization of gp4 when assembled with the portal ring. Furthermore, native and ion mobility mass spectrometry reveal a major conformational change in the portal upon binding of gp4. We propose that the gp4-induced conformational change in the portal ring initiates a cascade of events assisting in the stabilization of newly filled P22 particles, which marks the end of phage morphogenesis.

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Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4.

Publication Date: 2008 May 30 PMID: 18448122
Authors: Wong, J. H. - Fiala, K. A. - Suo, Z. - Ling, H.
Journal: J Mol Biol

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 A resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 A resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131 degrees rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.

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Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln.

Publication Date: 2008 May 30 PMID: 18448121
Authors: Dubin, G. - Stec-Niemczyk, J. - Kisielewska, M. - Pustelny, K. - Popowicz, G. M. - Bista, M. - Kantyka, T. - Boulware, K. T. - Stennicke, H. R. - Czarna, A. - Phopaisarn, M. - Daugherty, P. S. - Thogersen, I. B. - Enghild, J. J. - Thornberry, N. - Dubin, A. - Potempa, J.
Journal: J Mol Biol

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 A. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.

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Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.

Publication Date: 2008 May 30 PMID: 18448120
Authors: Xue, X. - Lehming, N.
Journal: J Mol Biol

Nhp6p is an architectural Saccharomyces cerevisiae non-histone chromosomal protein that bends DNA and plays an important role in transcription and genome stability. We used the split-ubiquitin system to isolate proteins that interact with Nhp6p in vivo, and we confirmed 11 of these protein-protein interactions with glutathione S-transferase pull-down experiments in vitro. Most of the Nhp6p-interacting proteins are involved in transcription and DNA repair. We utilized the ZDS1, PUR5 and UME6 genes, which are repressed by Nhp6p and its interacting partners Rpb4p and Med3p, to study the chromosomal localization of these three proteins in wild-type and gene deletion strains. Nhp6p, Med3p and Rpb4p were found at the promoters of ZDS1, PUR5 and UME6, indicating that the repressing effects the three proteins had on the expression of these three genes had been direct ones. We also found that Med3p inhibited promoter clearance of RNA polymerase II, which contained the dissociable subunit Rpb4p, while Nhp6p recruited Rpb4p to the basal promoters of ZDS1, PUR5 and UME6. Our results further suggest that Rpb4p inhibits transcription initiation but stimulates transcription elongation and that Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.

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A recruited protease is involved in catabolism of pyrimidines.

Publication Date: 2008 May 30 PMID: 18448119
Authors: Andersen, B. - Lundgren, S. - Dobritzsch, D. - Piskur, J.
Journal: J Mol Biol

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

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Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.

Publication Date: 2008 May 30 PMID: 18448118
Authors: Brouns, S. J. - Barends, T. R. - Worm, P. - Akerboom, J. - Turnbull, A. P. - Salmon, L. - van der Oost, J.
Journal: J Mol Biol

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

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Transcription factors GATA/ELT-2 and forkhead/HNF-3/PHA-4 regulate the tropomyosin gene expression in the pharynx and intestine of Caenorhabditis elegans.

Publication Date: 2008 May 30 PMID: 18448117
Authors: Anokye-Danso, F. - Anyanful, A. - Sakube, Y. - Kagawa, H.
Journal: J Mol Biol

Gene regulation during development is an important biological activity that leads to synthesis of biomolecules at specific locations and specific times. The single tropomyosin gene of Caenorhabditis elegans, tmy-1/lev-11, produces four isoforms of protein: two from the external promoter and two from the internal promoter. We investigated the internal promoter of tropomyosin to identify sequences that regulate expression of tmy-1 in the pharynx and intestine. By promoter deletion of tmy-1 reporters as well as by database analyses, a 100-bp fragment that contained binding sequences for a GATA factor, for a chicken CdxA homolog, and for a forkhead factor was identified. Both the forkhead and CdxA binding sequences contributed to pharyngeal and intestinal expression. In addition, the GATA site also influenced intestinal expression of tmy-1 reporter. We showed that ELT-2 and PHA-4 proteins interact directly with the GATA and forkhead binding sequences, respectively, in gel mobility shift assays. RNA interference knockdown of elt-2 diminished tmy-1::gfp expression in the intestine. In contrast to RNA interference knockdown of pha-4, expression of tmy-1::gfp in pha-4;smg-1 mutants was slightly weaker than that of the wild type. Ectopic expression of PHA-4 and ELT-2 by heat shock was sufficient to elicit widespread expression of tmy-1::lacZ reporter in embryos. We found no indication of a synergistic relation between ELT-2 and PHA-4. Based on our data, PHA-4 and CdxA function as general transcription factors for pharyngeal and intestinal regulation of tmy-1. We present models by which ELT-2, PHA-4, and CdxA orchestrate expression from the internal promoter of tmy-1.

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Hybridization between mitochondrial heavy strand tDNA and expressed light strand tRNA modulates the function of heavy strand tDNA as light strand replication origin.

Publication Date: 2008 May 23 PMID: 18440556
Authors: Seligmann, H.
Journal: J Mol Biol

Mitochondrial heavy strand (HS) tDNA codes for tRNAs and frequently functions as the light strand (LS) replication origin (OL). During replication, HS sites remain single-stranded until their LS complement is synthesized, a state prone to hydrolytic deaminations of C-->T and A-->G, causing genome-wide deamination gradients starting at OLs and proportional to time spent single-stranded. Gradient strength is proportional to OL formation by HS tDNAs. Hypothetically, hybridization between HS tDNA and its expressed complement tRNA should decrease OL activity for LS-, but not HS-encoded tRNAs. Comparisons between primate genomes and between pathogenic and non-pathogenic human polymorphisms both confirm corresponding predictions on OL activity. In primates, strengths of deamination gradients starting at tDNAs functioning as OLs and coding for LS tRNAs decrease proportionally to stabilities of HS tDNA-LS tRNA hybridization; not so for HS tRNAs. Similarly, in mutants of human HS tDNAs coding for LS tRNAs, pathogenic mutants of tDNAs usually not forming OLs form weaker HS tDNA-LS tRNA duplexes than non-pathogenic ones; the opposite is true for tDNAs usually forming OLs. No trend was detected for HS tDNA coding for HS tRNA. tDNA-tRNA hybridization of the modal (most frequent) human tDNA sequence is more stable than of other, rarer non-pathogenic polymorphisms, suggesting similar but weaker mutational effects on tDNA/tRNA functions than in pathogenic mutants. HS tDNA-LS tRNA hybridization appears to compete with OL formation by HS tDNA self-hybridization.

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Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit.

Publication Date: 2008 May 30 PMID: 18440555
Authors: Slade, D. J. - Lovelace, L. L. - Chruszcz, M. - Minor, W. - Lebioda, L. - Sodetz, J. M.
Journal: J Mol Biol

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that assemble on bacterial membranes to form a porelike structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8 alpha, C8 beta, C8 gamma) arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C6, C7 C8 alpha, C8 beta, and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8 gamma subunit is unrelated and belongs to the lipocalin family of proteins that display a beta-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8 alpha MACPF domain were recently reported and both display a fold similar to those of the bacterial pore-forming cholesterol-dependent cytolysins (CDCs). In the present study, we determined the crystal structure of the human C8 alpha MACPF domain disulfide-linked to C8 gamma (alphaMACPF-gamma) at 2.15 A resolution. The alphaMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8 gamma. One is in a previously characterized 19-residue insertion (indel) in C8 alpha and fills the entrance to the putative C8 gamma ligand-binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8 gamma beta-barrel. The latter interaction induces conformational changes in alphaMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X(6)-G-G in alphaMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.

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Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea.

Publication Date: 2008 May 23 PMID: 18440554
Authors: Walti, M. A. - Walser, P. J. - Thore, S. - Grunler, A. - Bednar, M. - Kunzler, M. - Aebi, M.
Journal: J Mol Biol

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.

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Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

Publication Date: 2008 May 30 PMID: 18440553
Authors: Chen, W. - Srinath, H. - Lam, S. S. - Schiffer, C. A. - Royer, W. E. Jr - Lin, K.
Journal: J Mol Biol

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP (amino acid residues 2067-2112) interacts directly with IRF-3 (173-427) and six of its single-site mutants to form heterodimers, but when CBP interacts with IRF-3 S396D, oligomerization is evident. CBP also interacts in vitro with IRF-3 double-site mutants to form different levels of oligomerization. Among all the single-site mutants, IRF-3 S396D showed the strongest binding to CBP. Although IRF-3 S386D alone did not interact as strongly with CBP as did other mutants, it strengthened the interaction and oligomerization of IRF-3 S396D with CBP. In contrast, IRF-3 S385D weakened the interaction and oligomerization of IRF-3 S396D and S386/396D with CBP. Thus, it appears that Ser385 and Ser386 serve antagonistic functions in regulating IRF-3 phosphoactivation. These results indicate that Ser386 and Ser396 are critical for IRF-3 activation, and support a phosphorylation-oligomerization model for IRF-3 activation.

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Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides.

Publication Date: 2008 May 23 PMID: 18440552
Authors: Epand, R. F. - Mowery, B. P. - Lee, S. E. - Stahl, S. S. - Lehrer, R. I. - Gellman, S. H. - Epand, R. M.
Journal: J Mol Biol

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.

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The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.

Publication Date: 2008 May 23 PMID: 18440551
Authors: Gasper, R. - Thomas, C. - Ahmadian, M. R. - Wittinghofer, A.
Journal: J Mol Biol

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.

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Folding kinetics of large RNAs.

Publication Date: 2008 May 23 PMID: 18440024
Authors: Geis, M. - Flamm, C. - Wolfinger, M. T. - Tanzer, A. - Hofacker, I. L. - Middendorf, M. - Mandl, C. - Stadler, P. F. - Thurner, C.
Journal: J Mol Biol

We introduce here a heuristic approach to kinetic RNA folding that constructs secondary structures by stepwise combination of building blocks. These blocks correspond to subsequences and their thermodynamically optimal structures. These are determined by the standard dynamic programming approach to RNA folding. Folding trajectories are modeled at base-pair resolution using the Morgan-Higgs heuristic and a barrier tree-based heuristic to connect combinations of the local building blocks. Implemented in the program Kinwalker, the algorithm allows co-transcriptional folding and can be used to fold sequences of up to about 1500 nucleotides in length. A detailed comparison with several well-studied examples from the literature, including the delayed folding of bacteriophage cloverleaf structures, the adenine sensing riboswitch, and the hok RNA, shows an excellent agreement of predicted trajectories and experimental evidence. The software is available as part of the ViennaRNA Package.

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Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway.

Publication Date: 2008 May 23 PMID: 18440023
Authors: Treiber, N. - Schulz, G. E.
Journal: J Mol Biol

The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 A resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2'-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed.

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Solution Conformation, backbone dynamics and lipid interactions of the intrinsically unstructured malaria surface protein MSP2.

Publication Date: 2008 May 23 PMID: 18440022
Authors: Zhang, X. - Perugini, M. A. - Yao, S. - Adda, C. G. - Murphy, V. J. - Low, A. - Anders, R. F. - Norton, R. S.
Journal: J Mol Biol

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little alpha-helix or beta-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.

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The Hsp90 inhibitor radicicol interacts with the ATP-binding pocket of bacterial sensor kinase PhoQ.

Publication Date: 2008 May 23 PMID: 18440021
Authors: Guarnieri, M. T. - Zhang, L. - Shen, J. - Zhao, R.
Journal: J Mol Biol

Sensor kinases in the bacterial two-component system share a unique ATP-binding Bergerat fold with the GHL (gyrase, Hsp90, and MutL) family of proteins. We demonstrated that selected GHL inhibitors bind to the catalytic domain of sensor kinase PhoQ (PhoQcat) using NMR chemical shift perturbation experiments. Using crystallographic approaches, we show that radicicol (an Hsp90 inhibitor) binds and interacts specifically with residues in the ATP-binding pocket of PhoQ. The interaction between radicicol and PhoQcat demonstrates significant similarities as well as differences compared to AMPPNP (a non-hydrolyzable ATP analog) bound to PhoQcat and radicicol bound to Hsp90. Our results suggest that GHL inhibitors may be useful lead compounds for developing sensor kinase inhibitors.

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Chiral bifurcation in aggregating insulin: an induced circular dichroism study.

Publication Date: 2008 May 23 PMID: 18439622
Authors: Loksztejn, A. - Dzwolak, W.
Journal: J Mol Biol

The structural unambiguity of folding is lost when disordered protein molecules convert into beta-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils' chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 degrees C, the bifurcation is no longer observed--fibrils' chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular beta-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged beta-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid "strains."

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Characterization of abasic endonuclease activity of human Ape1 on alternative substrates, as well as effects of ATP and sequence context on AP site incision.

Publication Date: 2008 May 23 PMID: 18439621
Authors: Berquist, B. R. - McNeill, D. R. - Wilson, D. M. 3rd
Journal: J Mol Biol

Human Ape1 is a multifunctional protein with a major role in initiating repair of apurinic/apyrimidinic (AP) sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone immediately adjacent to the damage. Besides in double-stranded DNA, Ape1 has been shown to cleave at AP sites in single-stranded regions of a number of biologically relevant DNA conformations and in structured single-stranded DNA. Extension of these studies has revealed a more expansive repertoire of model substrates on which Ape1 exerts AP endonuclease activity. In particular, Ape1 possesses the ability to cleave at AP sites located in (i) the DNA strand of a DNA/RNA hybrid, (ii) "pseudo-triplex" bubble substrates designed to mimic stalled replication or transcription intermediates, and (iii) configurations that emulate R-loop structures that arise during class switch recombination. Moreover, Ape1 was found to cleave AP-site-containing single-stranded RNA, suggesting a novel "cleansing" function that may contribute to the elimination of detrimental cellular AP-RNA molecules. Finally, sequence context immediately surrounding an abasic site in duplex DNA was found to have a less than threefold effect on the incision efficiency of Ape1, and ATP was found to exert complex effects on the endonuclease capacity of Ape1 on double-stranded substrates. The results suggest that in addition to abasic sites in conventional duplex genomic DNA, Ape1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and that Ape1 can endonucleolytically destroy damaged RNA.

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Zinc binding catalytic domain of human tankyrase 1.

Publication Date: 2008 May 23 PMID: 18436240
Authors: Lehtio, L. - Collins, R. - van den Berg, S. - Johansson, A. - Dahlgren, L. G. - Hammarstrom, M. - Helleday, T. - Holmberg-Schiavone, L. - Karlberg, T. - Weigelt, J.
Journal: J Mol Biol

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.

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Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex.

Publication Date: 2008 May 23 PMID: 18436239
Authors: Liu, C. - Li, D. - Liang, Y. H. - Li, L. F. - Su, X. D.
Journal: J Mol Biol

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the 'lid' motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the alpha-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.

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The nuclear I kappaB protein I kappaB zeta specifically binds NF-kappaB p50 homodimers and forms a ternary complex on kappaB DNA.

Publication Date: 2008 May 23 PMID: 18436238
Authors: Trinh, D. V. - Zhu, N. - Farhang, G. - Kim, B. J. - Huxford, T.
Journal: J Mol Biol

Although they share sequence homology to classical cytoplasmic I kappaB inhibitors of transcription factor NF-kappaB, the proteins I kappaB zeta, Bcl-3, and I kappa BNS function in the nucleus as factors that influence NF-kappaB-dependent gene expression profiles. Through the use of purified recombinant proteins and by comparison with the classical I kappaB protein I kappaB alpha, we have discovered mechanistic details of the interaction between I kappaB zeta and functional NF-kappaB dimers. Whereas I kappaB alpha and other classical I kappaB proteins bind tightly to NF-kappaB dimers that possess the p65 subunit, I kappaB zeta binds preferentially to NF-kappaB p50 homodimers. This altered specificity is particularly interesting in light of the fact that both NF-kappaB subunits exhibit high sequence and structural homology, while the I kappaB alpha and I kappaB zeta proteins are also conserved in primary amino acid sequence. We further show that I kappaB zeta is capable of forming a stable ternary complex with the NF-kappaB p50 homodimer and kappaB DNA. Again, this is a stark contrast from I kappaB alpha, which inhibits NF-kappaB p65 homodimer binding to NF-kappaB target DNA sequences. Removal of the DNA sequences flanking the NF-kappaB binding site does not directly affect the interaction of p50 and I kappaB zeta. Rather, we have discovered that the carboxy-terminal glycine-rich region of the NF-kappaB p50 homodimer is involved in mediating high-affinity binding of I kappaB zeta and NF-kappaB p50. We conclude that the NF-kappaB p50 homodimer functions as a legitimate activator of gene expression through formation of a ternary complex between itself, I kappaB zeta, and DNA. The requirement for formation of this complex could explain why the nuclear I kappaB protein I kappaB zeta is absolutely required for expression of the pluripotent pro-inflammatory cytokine interleukin-6 in peritoneal macrophages.

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Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition.

Publication Date: 2008 May 23 PMID: 18436237
Authors: Correia, M. A. - Prates, J. A. - Bras, J. - Fontes, C. M. - Newman, J. A. - Lewis, R. J. - Gilbert, H. J. - Flint, J. E.
Journal: J Mol Biol

The microbial degradation of the plant cell wall is of increasing industrial significance, exemplified by the interest in generating biofuels from plant cell walls. The majority of plant cell-wall polysaccharides are acetylated, and removal of the acetyl groups through the action of carbohydrate esterases greatly increases the efficiency of polysaccharide saccharification. Enzymes in carbohydrate esterase family 3 (CE3) are common in plant cell wall-degrading microorganisms but there is a paucity of structural and biochemical information on these biocatalysts. Clostridium thermocellum contains a single CE3 enzyme, CtCes3, which comprises two highly homologous (97% sequence identity) catalytic modules appended to a C-terminal type I dockerin that targets the esterase into the cellulosome, a large protein complex that catalyses plant cell wall degradation. Here, we report the crystal structure and biochemical properties of the N-terminal catalytic module (CtCes3-1) of CtCes3. The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. CtCes3-1 displays an alpha/beta hydrolase fold that contains a central five-stranded parallel twisted beta-sheet flanked by six alpha-helices. In addition, the enzyme contains a canonical catalytic triad in which Ser44 is the nucleophile, His208 is the acid-base and Asp205 modulates the basic nature of the histidine. The acetate moiety is accommodated in a hydrophobic pocket and the negative charge of the tetrahedral transition state is stabilized through hydrogen bonds with the backbone N of Ser44 and Gly95 and the side-chain amide of Asn124.

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Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1.

Publication Date: 2008 May 23 PMID: 18436236
Authors: Mantha, A. K. - Oezguen, N. - Bhakat, K. K. - Izumi, T. - Braun, W. - Mitra, S.
Journal: J Mol Biol

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3' blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg(2+). However, the Ser99 mutant has normal activity in 2 mM Mg(2+). Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg(2+), with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg(2+). This effect due to the mutation of Cys99, approximately 16 A from the active site, on the DNA binding and activity is surprising. Mg(2+) at the B site promotes stabilization of the C99S mutant complex. At higher Mg(2+) concentration the A site is also filled, causing the B-site Mg(2+) to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5' site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg(2+)]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.

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Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers.

Publication Date: 2008 May 16 PMID: 18433773
Authors: Trindade, M. B. - Job, V. - Contreras-Martel, C. - Pelicic, V. - Dessen, A.
Journal: J Mol Biol

Type IV pili (Tfp) are arguably the most widespread pili in bacteria, whose biogenesis requires a complex machinery composed of as many as 18 different proteins. This includes the conserved outer membrane-localized secretin, which forms a pore through which Tfp emerge on the bacterial surface. Although, in most model species studied, secretin oligomerization and functionality requires the action of partner lipoproteins, structural information regarding these molecules is limited. We report the high-resolution crystal structure of PilW, the partner lipoprotein of the type IV pilus secretin PilQ from Neisseria meningitidis, which defines a conserved class of Tfp biogenesis proteins involved in the formation and/or stability of secretin multimers in a wide variety of bacteria. The use of the PilW structure as a blueprint reveals an area of high-level sequence conservation in homologous proteins from different pathogens that could reflect a possible secretin-binding site. These results could be exploited for the development of new broad-spectrum antibacterials interfering with the biogenesis of a widespread virulence factor.

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Multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled alpha-synuclein.

Publication Date: 2008 May 16 PMID: 18433772
Authors: Thirunavukkuarasu, S. - Jares-Erijman, E. A. - Jovin, T. M.
Journal: J Mol Biol

The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson's disease as well as in basic investigations.

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The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution.

Publication Date: 2008 May 16 PMID: 18433771
Authors: Sukackaite, R. - Grazulis, S. - Bochtler, M. - Siksnys, V.
Journal: J Mol Biol

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5'-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-A resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5'-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

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Protein-protein interaction regulates proteins' mechanical stability.

Publication Date: 2008 May 16 PMID: 18433770
Authors: Cao, Y. - Yoo, T. - Zhuang, S. - Li, H.
Journal: J Mol Biol

Elastomeric proteins are molecular springs found not only in a variety of biological machines and tissues, but also in biomaterials of superb mechanical properties. Regulating the mechanical stability of elastomeric proteins is not only important for a range of biological processes, but also critical for the use of engineered elastomeric proteins as building blocks to construct nanomechanical devices and novel materials of well-defined mechanical properties. Here we demonstrate that protein-protein interactions can potentially serve as an effective means to regulate the mechanical properties of elastomeric proteins. We show that the binding of fragments of IgG antibody to a small protein, GB1, can significantly enhance the mechanical stability of GB1. The regulation of the mechanical stability of GB1 by IgG fragments is not through direct modification of the interactions in the mechanically key region of GB1; instead, it is accomplished via the long-range coupling between the IgG binding site and the mechanically key region of GB1. Although Fc and Fab bind GB1 at different regions of GB1, their binding to GB1 can increase the mechanical stability of GB1 significantly. Using alanine point mutants of GB1, we show that the amplitude of mechanical stability enhancement of GB1 by Fc does not correlate with the binding affinity, suggesting that binding affinity only affects the population of GB1/human Fc (hFc) complex at a given concentration of hFc, but does not affect the intrinsic mechanical stability of the GB1/hFc complex. Furthermore, our results indicate that the mechanical stability enhancement by IgG fragments is robust and can tolerate sequence/structural perturbation to GB1. Our results demonstrate that the protein-protein interaction is an efficient approach to regulate the mechanical stability of GB1-like proteins and we anticipate that this new methodology will help to develop novel elastomeric proteins with tunable mechanical stability and compliance.

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Molecular dynamics simulation of the Escherichia coli NikR protein: equilibrium conformational fluctuations reveal interdomain allosteric communication pathways.

Publication Date: 2008 May 16 PMID: 18433769
Authors: Bradley, M. J. - Chivers, P. T. - Baker, N. A.
Journal: J Mol Biol

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

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Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR.

Publication Date: 2008 May 16 PMID: 18423665
Authors: Hegedus, T. - Serohijos, A. W. - Dokholyan, N. V. - He, L. - Riordan, J. R.
Journal: J Mol Biol

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its approximately 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.

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Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation.

Publication Date: 2008 May 16 PMID: 18423664
Authors: Wu, K. P. - Kim, S. - Fela, D. A. - Baum, J.
Journal: J Mol Biol

Conversion of human alpha-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by (15)N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-A beta amyloid component (NAC) region that is both more exposed and has a higher propensity to beta structure may accelerate the rate of fibril formation of aS.

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The in vivo and in vitro aggregation properties of globular proteins correlate with their conformational stability: the SH3 case.

Publication Date: 2008 May 16 PMID: 18423663
Authors: Espargaro, A. - Castillo, V. - de Groot, N. S. - Ventura, S.
Journal: J Mol Biol

Protein misfolding and deposition underlie an increasing number of debilitating human disorders and constitute a problem of major concern in biotechnology. In the last years, in vitro studies have provided valuable insights into the physicochemical principles underlying protein aggregation. Nevertheless, information about the determinants of protein deposition within the cell is scarce and only a few systematic studies comparing in vitro and in vivo data have been reported. Here, we have used the SH3 domain of alpha-spectrin as a model globular protein in an attempt to understand the relationship between protein aggregation in the test-tube and in the more complex cellular environment. The investigation of the aggregation in Escherichia coli of this domain and a large set of mutants, together with the analysis of their sequential and conformational properties allowed us to evaluate the contribution of different polypeptidic factors to the cellular deposition of globular proteins. The data presented here suggest that the rules that govern in vitro protein aggregation are also valid in in vivo contexts. They also provide relevant insights into intracellular protein deposition in both conformational diseases and recombinant protein production.

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Porphyrin binding and distortion and substrate specificity in the ferrochelatase reaction: the role of active site residues.

Publication Date: 2008 May 16 PMID: 18423489
Authors: Karlberg, T. - Hansson, M. D. - Yengo, R. K. - Johansson, R. - Thorvaldsen, H. O. - Ferreira, G. C. - Hansson, M. - Al-Karadaghi, S.
Journal: J Mol Biol

The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation.

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Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six hypervariable loops.

Publication Date: 2008 May 16 PMID: 18423488
Authors: Laffly, E. - Pelat, T. - Cedrone, F. - Blesa, S. - Bedouelle, H. - Thullier, P.
Journal: J Mol Biol

The enhancement of antibody affinity by mutagenesis targeting only complementarity determining regions has the advantage of respecting the framework regions, which are important for tolerance if clinical use is envisaged. Here, starting from a Fab (antigen-binding fragment; 35PA(83)) capable of neutralizing the lethal toxin of anthrax and having an affinity of 3.4 nM for its antigen, a phage-displayed library of variants where all six complementarity determining regions (73 positions) were targeted for mutagenesis was built. This library contained 5 x 10(8) variants, and each variant carried four mutations on average. The library was first panned with adsorbed antigen and washes of increasing stringency. It was then screened in parallel with either small concentrations of soluble biotinylated antigen or adsorbed antigen and long elution times in the presence of soluble antigen. The stringencies of both selections were pushed as far as possible. Compared with 35PA(83), the best selected clone had an affinity enhanced 19-fold, to 180 pM, and its 50% inhibitory concentration was decreased by 40%. The results of the two selection methods were compared, and the generality of these methods was considered.

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Steric zipper of the amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein.

Publication Date: 2008 May 16 PMID: 18423487
Authors: Lee, S. W. - Mou, Y. - Lin, S. Y. - Chou, F. C. - Tseng, W. H. - Chen, C. H. - Lu, C. Y. - Yu, S. S. - Chan, J. C.
Journal: J Mol Biol

We report the results of atomic force microscopy, Fourier-transform infrared spectroscopy, solid-state nuclear magnetic resonance, and molecular dynamics (MD) calculations for amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein (H1). Our data reveal that H1 fibrils contain no more than two beta-sheet layers. The peptide strands of H1 fibrils are antiparallel with the A117 residues aligned to form a linear chain in the direction of the fibril axis. The molecular structure of the H1 fibrils, which adopts the motif of steric zipper, is highly uniform in the region of the palindrome sequence AGAAAAGA. The closest distance between the two adjacent beta-sheet layers is found to be about 5 A. The structural features of the molecular model of H1 fibrils obtained by MD simulations are consistent with the experimental results. Overall, our solid-state NMR and MD simulation data indicate that a steric zipper, which was first observed in the crystals of fibril-forming peptides, can be formed in H1 fibrils near the region of the palindrome sequence.

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NO sensing in Pseudomonas aeruginosa: structure of the transcriptional regulator DNR.

Publication Date: 2008 May 16 PMID: 18420222
Authors: Giardina, G. - Rinaldo, S. - Johnson, K. A. - Di Matteo, A. - Brunori, M. - Cutruzzola, F.
Journal: J Mol Biol

All denitrifying bacteria can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels, controlling the expression of the denitrification gene clusters by redox signaling, mainly through transcriptional regulators belonging either to the DNR (dissimilative nitrate respiration regulator) or to the NnrR (nitrite and nitric oxide reductase regulator) subgroups of the FNR (fumarate and nitrate reductase regulatory protein)-CRP (cAMP receptor protein) superfamily. The NO dependence of the transcriptional activity of promoters regulated by these transcription factors has suggested that they may act as NO sensors in vivo. Despite great interest in the regulation of denitrification, which in Pseudomonas aeruginosa is strictly related to virulence, functional and structural characterization of these NO sensors is still lacking. Here we present the three-dimensional structure of the sensor domain of the DNR from P. aeruginosa at 2.1 A resolution. This is the first structure of a putative NO-sensing bacterial transcriptional regulator and reveals the presence of a large hydrophobic cavity that may be the cofactor binding site. Parallel spectroscopic evidence indicates that apo-DNR binds heme in vitro and that the heme-bound form reacts with carbon monoxide and NO, thus supporting the hypothesis that NO sensing involves gas binding to the ferrous heme. Preliminary experiments indicate that heterologous expression of the heme-containing DNR yields a protein able to bind DNA in vitro.

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Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain.

Publication Date: 2008 May 16 PMID: 18407291
Authors: Shimojo, H. - Sano, N. - Moriwaki, Y. - Okuda, M. - Horikoshi, M. - Nishimura, Y.
Journal: J Mol Biol

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded beta-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional beta-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.

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Interaction of the tylosin-resistance methyltransferase RlmA II at its rRNA target differs from the orthologue RlmA I.

Publication Date: 2008 May 16 PMID: 18406425
Authors: Douthwaite, S. - Jakobsen, L. - Yoshizawa, S. - Fourmy, D.
Journal: J Mol Biol

RlmA(II) methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-producer Streptomyces fradiae and the pathogen Streptococcus pneumoniae. Recombinant S. pneumoniae RlmA(II) was purified and shown to retain its activity and specificity in vitro when tested on unmethylated 23 S rRNA substrates. RlmA(II) makes multiple footprint contacts with nucleotides in stem-loops 33, 34 and 35, and does not interact elsewhere in the rRNA. Binding of RlmA(II) to the rRNA is dependent on the cofactor S-adenosylmethionine (or S-adenosylhomocysteine). RlmA(II) interacts with the same rRNA region as the orthologous enzyme RlmA(I) that methylates at nucleotide G745. Differences in nucleotide contacts within hairpin 35 indicate how the two methyltransferases recognize their distinct targets.

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Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin.

Publication Date: 2008 May 16 PMID: 18406424
Authors: Fernandes, H. - Pasternak, O. - Bujacz, G. - Bujacz, A. - Sikorski, M. M. - Jaskolski, M.
Journal: J Mol Biol

Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel beta-sheet and a long C-terminal alpha-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 A resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.

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A unique mode of microtubule stabilization induced by peloruside A.

Publication Date: 2008 May 16 PMID: 18405918
Authors: Huzil, J. T. - Chik, J. K. - Slysz, G. W. - Freedman, H. - Tuszynski, J. - Taylor, R. E. - Sackett, D. L. - Schriemer, D. C.
Journal: J Mol Biol

Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the alpha/beta-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of beta-tubulin at a previously unknown ligand site, rather than on alpha-tubulin as suggested in earlier studies.

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Integrin alpha IIb beta 3 in a membrane environment remains the same height after Mn2+ activation when observed by cryoelectron tomography.

Publication Date: 2008 May 16 PMID: 18405917
Authors: Ye, F. - Liu, J. - Winkler, H. - Taylor, K. A.
Journal: J Mol Biol

Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using cryoelectron tomography, we examined the simplest predication of the different integrin activation models, i.e., the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn(2+)-induced activation of membrane-bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.

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