Journal of Molecular Biology

Structural Properties of EGCG-Induced, Nontoxic Alzheimer's Disease Abeta Oligomers.

Publication Date: 2012 Jan 28 PMID: 22300765
Authors: Lopez Del Amo, J. M. - Fink, U. - Dasari, M. - Grelle, G. - Wanker, E. E. - Bieschke, J. - Reif, B.
Journal: J Mol Biol

The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease beta-amyloid peptide (Abeta) neurotoxicity. Solution-state NMR allows probing initial EGCG-Abeta interactions. We show that EGCG-induced Abeta oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Abeta. The C-terminal part of the Abeta peptide (residues 22-39) adopts a beta-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Abeta aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.

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CNP-1 (ARRD-17), a Novel Substrate of Calcineurin, Is Critical for Modulation of Egg-Laying and Locomotion in Response to Food and Lysine Sensation in Caenorhabditis elegans.

Publication Date: 2012 Jan 28 PMID: 22300764
Authors: Jee, C. - Choi, T. W. - Kalichamy, K. - Yee, J. Z. - Song, H. O. - Ji, Y. J. - Lee, J. - Lee, J. - L'etoile, N. - Ahnn, J. - Lee, S. K.
Journal: J Mol Biol

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.

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Structural Basis of Substrate Binding Specificity Revealed by the Crystal Structures of Polyamine Receptors SpuD and SpuE from Pseudomonas aeruginosa.

Publication Date: 2012 Jan 28 PMID: 22300763
Authors: Wu, D. - Lim, S. C. - Dong, Y. - Wu, J. - Tao, F. - Zhou, L. - Zhang, L. H. - Song, H.
Journal: J Mol Biol

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a key virulence determinant whose expression is induced by polyamine signals from mammalian host. SpuD and SpuE were postulated to be spermidine-preferential binding proteins, which regulate the polyamine content in this bacterial pathogen. In this study, we found that SpuD is a putrescine-preferential binding protein, while SpuE binds to spermidine exclusively. We have determined the crystal structures of SpuD in free form and in complex with putrescine and SpuE in free form and in complex with spermidine. Upon ligand binding, SpuD and SpuE undergo an "open-to-closed" conformational switch with the resultant closed ligand-bound forms, SpuD-putrescine and SpuE-spermidine, similar to their Escherichia coli counterparts PotF-putrescine and PotD-spermidine, respectively. Structural comparison suggested that two aromatic residues, Trp271 of SpuE and Phe273 of SpuD in segment II region, are the key structural determinants for putrescine/spermidine recognition specificity. Mutagenesis combined with isothermal titration calorimetry showed that substitution of Trp271 by Phe enabled SpuE gain substantial binding affinity for putrescine, while replacement of Phe273 by Trp reduced the binding affinity of SpuD toward putrescine by 250-fold. Altogether, these results revealed the molecular mechanism governing polyamine recognition specificity by SpuD and SpuE and provide the basis for further structural and functional studies of polyamine signal importation system in P. aeruginosa.

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Crystal Structure of the Urokinase Receptor in a Ligand-Free Form.

Publication Date: 2012 Jan 21 PMID: 22285761
Authors: Xu, X. - Gardsvoll, H. - Yuan, C. - Lin, L. - Ploug, M. - Huang, M.
Journal: J Mol Biol

The urokinase receptor urokinase-type plasminogen activator receptor (uPAR) is a surface receptor capable of not only focalizing urokinase-type plasminogen activator (uPA)-mediated fibrinolysis to the pericellular micro-environment but also promoting cell migration and chemotaxis. Consistent with this multifunctional role, uPAR binds several extracellular ligands, including uPA and vitronectin. Structural studies suggest that uPAR possesses structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 A and in complex with amino-terminal fragment (ATF) to 3.2 A. The structure of uPAR(H47C/N259C) in complex with ATF resembles the wild-type uPAR.ATF complex, demonstrating that these mutations do not perturb the uPA binding properties of uPAR. The present structure of uPAR(H47C/N259C) provides the first structural definition of uPAR in its ligand-free form, which represents one of the biologically active conformations of uPAR as defined by extensive biochemical studies. The domain boundary between uPAR DI-DII domains is more flexible than the DII-DIII domain boundary. Two important structural features are highlighted by the present uPAR structure. First, the DI-DIII domain boundary may face the cell membrane. Second, loop 130-142 of uPAR plays a dynamic role during ligand loading/unloading. Together, these studies provide new insights into uPAR structure-function relationships, emphasizing the importance of the inter-domain dynamics of this modular receptor.

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Protein Structure Determination from Pseudocontact Shifts Using ROSETTA.

Publication Date: 2012 Jan 18 PMID: 22285518
Authors: Schmitz, C. - Vernon, R. - Otting, G. - Baker, D. - Huber, T.
Journal: J Mol Biol

Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Deltachi-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Deltachi-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge on the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.

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Evidence for the Existence of a Secondary Pathway for Fibril Growth during the Aggregation of Tau.

Publication Date: 2012 Jan 17 PMID: 22281439
Authors: Ramachandran, G. - Udgaonkar, J. B.
Journal: J Mol Biol

The mechanism of amyloid fibril formation by proteins has been classically described by the nucleation-dependent polymerization (NDP) model, which makes certain predictions regarding the kinetics of fibrillation. All proteins whose aggregation conforms to the NDP model display a t(2) time dependence for their initial reaction profile. However, there are proteins whose aggregation reactions have kinetic signatures of a flat lag phase followed by an exponential rise in fibril mass, which does not conform to the NDP model. Amyloid fibril formation by tau, a microtubule-associated protein whose aggregation to form neurofibrillary tangles is implicated in Alzheimer's disease and other tauopathies, in the presence of inducers such as heparin and fatty acid micelles, has always been traditionally described by a ligand-induced NDP model. In this study, the existence of a secondary pathway for fibril growth during the aggregation of the functional, repeat domain of tau in the presence of heparin has been established. Both kinetic and accessory evidence are provided for the existence of this pathway, which is shown to augment the primary homogeneous nucleation pathway. From the kinetic data, the main secondary pathway that is operative appears to be fibril fragmentation but other pathways such as branching or secondary nucleation may also be operative.

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Molecular Interactions of Alzheimer's Abeta Protofilaments with Lipid Membranes.

Publication Date: 2012 Jan 17 PMID: 22281438
Authors: Tofoleanu, F. - Buchete, N. V.
Journal: J Mol Biol

Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid beta (Abeta) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Abeta fibrils and their adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Abeta protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic beta-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Abeta charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.

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Catalytic Activity and Acyl-Chain Selectivity of Diacylglycerol Kinase varepsilon Are Modulated by Residues in and near the Lipoxygenase-Like Motif.

Publication Date: 2012 Jan 12 PMID: 22266092
Authors: D'Souza, K. - Epand, R. M.
Journal: J Mol Biol

Diacylglycerol kinase (DGK) varepsilon plays an important role in the resynthesis of phosphatidylinositol by mediating the phosphorylation of diacylglycerol to phosphatidic acid. DGKvarepsilon is unique among mammalian DGK isoforms in that it is the only one that shows acyl-chain selectivity, preferring diacylglycerols with an sn-2 arachidonoyl group. The region responsible for this arachidonoyl specificity is the lipoxygenase (LOX)-like motif found in the accessory domain, adjacent to DGKvarepsilon's catalytic site. Many mutations within the LOX-like motif result in a loss of enzyme activity. However, the few mutants that retain significant activity exhibit some decrease in selectivity for the arachidonoyl chain. In the present work, we have explored mutations in a region adjacent to the LOX-like motif, which is also contained within the same hydrophobic segment of the protein. This adjacent region also contains a cholesterol recognition/interaction amino acid consensus motif. Being outside of the LOX-like motif, this region likely has less direct contact with the substrate, and more activity is retained with mutations. This has allowed us to probe in more detail the relationship between this region of the protein and substrate specificity. We demonstrate that this cholesterol recognition/interaction amino acid consensus domain also plays a role in acyl-chain selectivity. Despite the high degree of conservation of the amino acid sequence in this region of the protein, certain mutations result in proteins with higher activity than the wild-type protein. These mutations also result in a selective gain of acyl-chain preferences for diacylglycerols with different acyl-chain profiles. In addition to the LOX-like motif, adjacent residues also contribute to selectivity for diacylglycerols with specific acyl-chain compositions, such as those found in the phosphatidylinositol cycle.

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Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32.

Publication Date: 2012 Jan 10 PMID: 22261232
Authors: Pavlova, O. - Lavysh, D. - Klimuk, E. - Djordjevic, M. - Ravcheev, D. A. - Gelfand, M. S. - Severinov, K. - Akulenko, N.
Journal: J Mol Biol

Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase (RNAP): gp79, a novel protein, and gp36, a distant homolog of sigma(70) family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during infection. Host transcription shutoff and three distinct bacteriophage temporal gene classes (early, middle, and late) were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by a host RNAP holoenzyme containing the sigma(70) factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by an RNAP holoenzyme containing gp36. These promoters are characterized by a single-consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by sigma(70) holoenzyme, activated transcription by the gp36 holoenzyme in vitro.

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Roles of Extracellular Chaperones in Amyloidosis.

Publication Date: 2012 Jan 10 PMID: 22248589
Authors: Wyatt, A. R. - Yerbury, J. J. - Dabbs, R. A. - Wilson, M. R.
Journal: J Mol Biol

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

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Unperturbing a Non-Helically Perturbed Bacterial Flagellar Filament: Salmonella typhimurium SJW23.

Publication Date: 2012 Jan 10 PMID: 22248588
Authors: Nisani-Bizer, K. - Trachtenberg, S.
Journal: J Mol Biol

Salmonella typhimurium SJW23 has a right-handed, non-helically perturbed filament of serotype gt with a unique surface pattern. Non-helical perturbations involve symmetry reduction along the five-start helical lines resulting in layer lines of fractional Bessel orders and a consequent seam. The flagellin gene, fliC(23), which we sequenced, differs from the sequence of the canonic, plain SJW1655 flagellin, fliC(1655). We modified discrete components of fliC(23) in order to localize, in the expressed filament, the submolecular site responsible for the non-helical perturbation. These modifications include (i) deleting the outermost domain D3(23), (ii) replacing D3(23) with D3(1655), (iii) substituting a hydrophilic alpha-helix at the interface between the neighboring domains D1 and D2 with a hydrophobic one from fliC(1655), and (iv) substituting a serine/glycine pair in the loop connecting the modified alpha-helix to its neighbor; these modifications were made in the presence and absence of D3(23). We used S. typhimurium SJW1655 both as a reference and as a source for 'spare parts'. The symmetry of the constructs was assessed from the power spectra through changes in the layer lines at a height of 1/105 and 1/35 A(-1), unique to the non-helical perturbation. Deleting D3(23), either alone or in combination with various substitutions, or replacing it with D3(1655) transforms the non-helically perturbed filament into a plain one as judged by the disappearance of the typical layer lines from the power spectra. We conclude that the non-helical perturbation is a product of unique interactions in the D3(23) density shell. Whereas other minor structural changes may occur at the filaments interior, they are all helically symmetric.

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Oligomeric Intermediates in Amyloid Formation: Structure Determination and Mechanisms of Toxicity.

Publication Date: 2012 Jan 12 PMID: 22248587
Authors: Fandrich, M.
Journal: J Mol Biol

Oligomeric intermediates are non-fibrillar polypeptide assemblies that occur during amyloid fibril formation and that are thought to underlie the aetiology of amyloid diseases, such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Focusing primarily on the oligomeric states formed from Alzheimer's disease beta-amyloid (Abeta) peptide, this review will make references to other polypeptide systems, highlighting common principles or sequence-specific differences. The covered topics include the structural properties and polymorphism of oligomers, the biophysical mechanism of peptide self-assembly and its role for pathogenicity in amyloid disease. Oligomer-dependent toxicity mechanisms will be explained along with recently emerging possibilities of interference.

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From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.

Publication Date: 2012 Jan 8 PMID: 22245580
Authors: Ohlendorf, R. - Vidavski, R. R. - Eldar, A. - Moffat, K. - Moglich, A.
Journal: J Mol Biol

Signaling photoreceptors mediate diverse organismal adaptations in response to light. As light-gated protein switches, signaling photoreceptors provide the basis for optogenetics, a term that refers to the control of organismal physiology and behavior by light. We establish as novel optogenetic tools the plasmids pDusk and pDawn, which employ blue-light photoreceptors to confer light-repressed or light-induced gene expression in Escherichia coli with up to 460-fold induction upon illumination. Key features of these systems are low background activity, high dynamic range, spatial control on the 20-mum scale, independence from exogenous factors, and ease of use. In optogenetic experiments, pDusk and pDawn can be used to specifically perturb individual nodes of signaling networks and interrogate their role. On the preparative scale, pDawn can induce by light the production of recombinant proteins and thus represents a cost-effective and readily automated alternative to conventional induction systems.

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Malleability of the Folding Mechanism of the Outer Membrane Protein PagP: Parallel Pathways and the Effect of Membrane Elasticity.

Publication Date: 2012 Jan 8 PMID: 22245579
Authors: Huysmans, G. H. - Radford, S. E. - Baldwin, S. A. - Brockwell, D. J.
Journal: J Mol Biol

Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC(12:0)PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower folding rate constant is observed. Interrupted refolding assays demonstrated that the two exponential phases reflect the presence of parallel folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC(12:0)PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC(12:0)PC liposomes resulted in a decrease in the folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the folding mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing folding routes to a unique native structure.

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Complex Spatial Organization and Flagellin Composition of Flagellar Propeller from Marine Magnetotactic Ovoid Strain MO-1.

Publication Date: 2012 Jan 5 PMID: 22245577
Authors: Zhang, W. J. - Santini, C. L. - Bernadac, A. - Ruan, J. - Zhang, S. D. - Kato, T. - Li, Y. - Namba, K. - Wu, L. F.
Journal: J Mol Biol

Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 mum/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an approximately 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity.

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Non-Stressful Death of 23S rRNA Mutant G2061C Defective in Puromycin Reaction.

Publication Date: 2012 Jan 10 PMID: 22245576
Authors: Sergiev, P. V. - Lesnyak, D. V. - Burakovsky, D. E. - Svetlov, M. - Kolb, V. A. - Serebryakova, M. V. - Demina, I. A. - Govorun, V. M. - Dontsova, O. A. - Bogdanov, A. A.
Journal: J Mol Biol

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.

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Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding.

Publication Date: 2012 Jan 8 PMID: 22245575
Authors: Ha, J. H. - Karchin, J. M. - Walker-Kopp, N. - Huang, L. S. - Berry, E. A. - Loh, S. N.
Journal: J Mol Biol

Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the "mutually exclusive folding" design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-A amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-A X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate "smart" biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.

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Propagation of the Prion Phenomenon: Beyond the Seeding Principle.

Publication Date: 2012 Jan 5 PMID: 22245492
Authors: Munch, C. - Bertolotti, A.
Journal: J Mol Biol

The deposition of misfolded proteins is the hallmark of the late-onset, rapidly progressive and devastating neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These diseases are caused by a gain of toxic properties associated with the propensity of otherwise soluble proteins to misfold. What governs the deposition of the disease-causing proteins in aged neurons is unclear, but recent evidence suggests that once misfolded, the diverse proteins associated with the neurodegenerative diseases can induce aggregation of their soluble counterpart, thereby sharing one of the defining properties of prions. In addition to the seeded polymerization, prions have the ability to replicate their aberrant conformation indefinitely and are transmissible. Are these properties also shared by diverse misfolded proteins?

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Inhibition of Amyloid Formation.

Publication Date: 2012 Jan 5 PMID: 22244855
Authors: Hard, T. - Lendel, C.
Journal: J Mol Biol

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on amyloid inhibition and clearance.

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The Transthyretin Amyloidoses: From Delineating the Molecular Mechanism of Aggregation Linked to Pathology to a Regulatory-Agency-Approved Drug.

Publication Date: 2012 Jan 5 PMID: 22244854
Authors: Johnson, S. M. - Connelly, S. - Fearns, C. - Powers, E. T. - Kelly, J. W.
Journal: J Mol Biol

Transthyretin (TTR) is one of the many proteins that are known to misfold and aggregate (i.e., undergo amyloidogenesis) in vivo. The process of TTR amyloidogenesis causes nervous system and/or heart pathology. While several of these maladies are associated with mutations that destabilize the native TTR quaternary and/or tertiary structure, wild-type TTR amyloidogenesis also leads to the degeneration of postmitotic tissue. Over the past 20 years, much has been learned about the factors that influence the propensity of TTR to aggregate. This biophysical information led to the development of a therapeutic strategy, termed "kinetic stabilization," to prevent TTR amyloidogenesis. This strategy afforded the drug tafamidis which was recently approved by the European Medicines Agency for the treatment of TTR familial amyloid polyneuropathy, the most common familial TTR amyloid disease. Tafamidis is the first and currently the only medication approved to treat TTR familial amyloid polyneuropathy. Here we review the biophysical basis for the kinetic stabilization strategy and the structure-based drug design effort that led to this first-in-class pharmacologic agent.

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A Cyclic Peptide Inhibitor of ApoC-II Peptide Fibril Formation: Mechanistic Insight from NMR and Molecular Dynamics Analysis.

Publication Date: 2012 Jan 5 PMID: 22244853
Authors: Griffin, M. D. - Yeung, L. - Hung, A. - Todorova, N. - Mok, Y. F. - Karas, J. A. - Gooley, P. R. - Yarovsky, I. - Howlett, G. J.
Journal: J Mol Biol

The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation.

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Arginine Changes the Conformation of the Arginine Attenuator Peptide Relative to the Ribosome Tunnel.

Publication Date: 2012 Jan 5 PMID: 22244852
Authors: Wu, C. - Wei, J. - Lin, P. J. - Tu, L. - Deutsch, C. - Johnson, A. E. - Sachs, M. S.
Journal: J Mol Biol

The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.

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Subcellular Distribution of the Human Putative Nucleolar GTPase GNL1 is Regulated by a Novel Arginine/Lysine-Rich Domain and a GTP Binding Domain in a Cell Cycle-Dependent Manner.

Publication Date: 2012 Jan 5 PMID: 22244851
Authors: Boddapati, N. - Anbarasu, K. - Suryaraja, R. - Tendulkar, A. V. - Mahalingam, S.
Journal: J Mol Biol

GNL1, a putative nucleolar GTPase, belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. Members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization with respect to cell growth remain unknown. To understand the nuclear transport mechanism of GNL1, we have identified a novel arginine/lysine-rich nucleolar localization signal in the NH(2)-terminus that is shown to translocate GNL1 and a heterologous protein to the nucleus/nucleolus in a pathway that is independent of importin-alpha and importin-beta. In addition, the present investigation provided evidence that GNL1 localized to the nucleus and the nucleolus only in G2 stage, in contrast to its cytoplasmic localization in the G1 and S phases of the cell cycle. Using heterokaryon assay, we have demonstrated that GNL1 shuttles between the nucleus and the cytoplasm and that the motif between amino acids 201 and 225 is essential for its export from the nucleus by a signal-mediated CRM1-independent pathway. Alanine-scanning mutagenesis of conserved residues within G-domains suggests that the G2 motif is critical for guanine nucleotide triphosphate (GTP) binding of GNL1 and further showed that nucleolar retention of GNL1 is regulated by a GTP-gating-mediated mechanism. Expression of wild-type GNL1 promotes G2/M transition, in contrast to the G-domain mutant (G2m), which fails to localize to the nucleolus. These data suggest that nucleolar translocation during G2 phase may be critical for faster M-phase transition during cell proliferation. Replacement of conserved residues within the G5 motif alters the stability of GNL1 without changing GTP binding activity. Finally, our data suggest that ongoing transcription is essential for the efficient localization of GNL1 to the nucleolus. Overall, the results reported here demonstrate that multiple mechanisms are involved in the translocation of GNL1 to the nucleolus in a cell cycle-dependent manner to regulate cell growth and proliferation.

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Evolutionary Walk between (beta/alpha)(8) Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase.

Publication Date: 2012 Feb 17 PMID: 22226942
Authors: Saab-Rincon, G. - Olvera, L. - Olvera, M. - Rudino-Pinera, E. - Benites, E. - Soberon, X. - Morett, E.
Journal: J Mol Biol

The functionally versatile (beta/alpha)(8) barrel scaffold was used to migrate triosephosphate isomerase (TPI) to thiamin phosphate synthase (TPS) activity, two enzymes that share the same fold but catalyze unrelated reactions through different mechanisms. The high sensitivity of the selection methodology was determinant to succeed in finding proteins with the desired activity. A combination of rational design and random mutagenesis was used to achieve the desired catalytic migration. One of the parallel directed evolution strategies followed resulted in TPI derivatives able to complement the thiamin phosphate auxotrophy phenotype of an Escherichia coli strain deleted of thiE, the gene that codes for TPS. Successive rounds of directed evolution resulted in better complementing TPI variants. Biochemical characterization of some of the evolved TPI clones demonstrated that the K(m) for the TPS substrates was similar to that of the native TPS; however and in agreement with the very slow complementation phenotype, the k(cat) was 4 orders of magnitude lower, indicating that substrate binding played a major role on selection. Interestingly, the crystal structure of the most proficient variant showed a slightly modified TPI active site occupied by a thiamin-phosphate-like molecule. Substitution of key residues in this region reduced TPS activity, strongly suggesting that this is also the catalytic site for the evolved TPS activity. The presence of the TPS reaction product at the active site explains the fast inactivation of the enzyme observed. In conclusion, by combining rational design, random mutagenesis and a very sensitive selection, it is possible to achieve enzymatic activity migration.

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Crystal Structure and Regulation Mechanisms of the CyaB Adenylyl Cyclase from the Human Pathogen Pseudomonas aeruginosa.

Publication Date: 2012 Feb 17 PMID: 22226839
Authors: Topal, H. - Fulcher, N. B. - Bitterman, J. - Salazar, E. - Buck, J. - Levin, L. R. - Cann, M. J. - Wolfgang, M. C. - Steegborn, C.
Journal: J Mol Biol

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a major cause of healthcare-associated infections. While the organism's intrinsic and acquired resistance to most antibiotics hinders treatment of P. aeruginosa infections, the regulatory networks controlling its virulence provide novel targets for drug development. CyaB, a key regulator of P. aeruginosa virulence, belongs to the Class III adenylyl cyclase (AC) family of enzymes that synthesize the second messenger cyclic adenosine 3',5'-monophosphate. These enzymes consist of a conserved catalytic domain fused to one or more regulatory domains. We describe here the biochemical and structural characterization of CyaB and its inhibition by small molecules. We show that CyaB belongs to the Class IIIb subfamily, and like other subfamily members, its activity is stimulated by inorganic carbon. CyaB is also regulated by its N-terminal MASE2 (membrane-associated sensor 2) domain, which acts as a membrane anchor. Using a genetic screen, we identified activating mutations in CyaB. By solving the crystal structure of the CyaB catalytic domain, we rationalized the effects of these mutations and propose that CyaB employs regulatory mechanisms similar to other Class III ACs. The CyaB structure further indicates subtle differences compared to other Class III ACs in both the active site and the inhibitor binding pocket. Consistent with these differences, we observed a unique inhibition profile, including identification of a CyaB selective compound. Overall, our results reveal mechanistic details of the physiological and pharmacological regulation of CyaB and provide the basis for its exploitation as a therapeutic drug target.

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Conformational properties of the unfolded state of im7 in nondenaturing conditions.

Publication Date: 2012 Feb 17 PMID: 22226836
Authors: Pashley, C. L. - Morgan, G. J. - Kalverda, A. P. - Thompson, G. S. - Kleanthous, C. - Radford, S. E.
Journal: J Mol Biol

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A-L19A-L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, (15)N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A-L19A-L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R(h)=25 A) relative to the urea-denatured state (R(h)>/=30 A) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.

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N-Terminal Segments Modulate the alpha-Helical Propensities of the Intrinsically Disordered Basic Regions of bZIP Proteins.

Publication Date: 2012 Feb 17 PMID: 22226835
Authors: Das, R. K. - Crick, S. L. - Pappu, R. V.
Journal: J Mol Biol

Basic region leucine zippers (bZIPs) are modular transcription factors that play key roles in eukaryotic gene regulation. The basic regions of bZIPs (bZIP-bRs) are necessary and sufficient for DNA binding and specificity. Bioinformatic predictions and spectroscopic studies suggest that unbound monomeric bZIP-bRs are uniformly disordered as isolated domains. Here, we test this assumption through a comparative characterization of conformational ensembles for 15 different bZIP-bRs using a combination of atomistic simulations and circular dichroism measurements. We find that bZIP-bRs have quantifiable preferences for alpha-helical conformations in their unbound monomeric forms. This helicity varies from one bZIP-bR to another despite a significant sequence similarity of the DNA binding motifs (DBMs). Our analysis reveals that intramolecular interactions between DBMs and eight-residue segments directly N-terminal to DBMs are the primary modulators of bZIP-bR helicities. We test the accuracy of this inference by designing chimeras of bZIP-bRs to have either increased or decreased overall helicities. Our results yield quantitative insights regarding the relationship between sequence and the degree of intrinsic disorder within bZIP-bRs, and might have general implications for other intrinsically disordered proteins. Understanding how natural sequence variations lead to modulation of disorder is likely to be important for understanding the evolution of specificity in molecular recognition through intrinsically disordered regions (IDRs).

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Energy complexes are apparently associated with the switch-motor complex of bacterial flagella.

Publication Date: 2012 Feb 17 PMID: 22210351
Authors: Zarbiv, G. - Li, H. - Wolf, A. - Cecchini, G. - Caplan, S. R. - Sourjik, V. - Eisenbach, M.
Journal: J Mol Biol

Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F(0)F(1) ATP synthase via its beta subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the beta subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F(0)F(1) ATP synthase with FliG is important for the function of the switch of bacterial flagella.

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Nucleocapsid Protein Annealing of a Primer-Template Enhances (+)-Strand DNA Synthesis and Fidelity by HIV-1 Reverse Transcriptase.

Publication Date: 2012 Feb 3 PMID: 22210155
Authors: Kim, J. - Roberts, A. - Yuan, H. - Xiong, Y. - Anderson, K. S.
Journal: J Mol Biol

Human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer, which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Through the use of a pre-steady-state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3- to 7-fold) in the rate of incorporation (k(pol)) by RT as compared to heat-annealed primer-template with single-nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (K(d)) of the nucleotide. These differences in k(pol) and K(d) were not through direct interactions between HIV-1 RT and NCp7. When extension by RT was examined, the data suggest that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat-annealed primer-template. This enhancement in rate is mediated through interactions with NCp7's zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV.

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Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme.

Publication Date: 2012 Feb 17 PMID: 22210154
Authors: Vaaje-Kolstad, G. - Bohle, L. A. - Gaseidnes, S. - Dalhus, B. - Bjoras, M. - Mathiesen, G. - Eijsink, V. G.
Journal: J Mol Biol

Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase (ef0361; EfChi18A) and a family 33 CBM (carbohydrate binding module) (ef0362; EfCBM33A) that catalyze chitin conversion in vitro. Various types of enzyme activity assays showed that EfChi18A has functional properties characteristic of an endochitinase. EfCBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of EfCBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that EfCBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens. Metal-depletion studies showed that EfCBM33A is a copper enzyme. In the presence of an external electron donor, EfCBM33A boosted the activity of EfChi18A, and combining the two enzymes led to rapid and complete conversion of beta-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field.

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Key structural features of the actin filament arp2/3 complex branch junction revealed by molecular simulation.

Publication Date: 2012 Feb 10 PMID: 22206989
Authors: Pfaendtner, J. - Volkmann, N. - Hanein, D. - Dalhaimer, P. - Pollard, T. D. - Voth, G. A.
Journal: J Mol Biol

We investigated the structure, properties and dynamics of the actin filament branch junction formed by actin-related protein (Arp) 2/3 complex using all-atom molecular dynamics (MD) simulations based on a model fit to a reconstruction from electron tomograms. Simulations of the entire structure consisting of 31 protein subunits together with solvent molecules containing approximately 3 million atoms were performed for an aggregate time of 175 ns. One 75-ns simulation of the original reconstruction was compared to two 50-ns simulations of alternate structures, showing that the hypothesized branch junction structure is very stable. Our simulations revealed that the interface between Arp2/3 complex and the mother actin filament features a large number of salt bridges and hydrophobic contacts, many of which are dynamic and formed/broken on the timescale of the simulation. The simulations suggest that the DNase binding loops in Arp3, and possibly Arp2, form stabilizing contacts with the mother filament. Unbiased comparison of models sampled from the MD simulation trajectory with the primary experimental electron tomography data identified regions were snapshots from the simulation provide atomic details of the model structures and also pinpoints regions where the initial modeling based on the electron tomogram reconstruction may be suboptimal.

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Molecular Basis of the Functional Divergence of Fatty Acyl-AMP Ligase Biosynthetic Enzymes of Mycobacterium tuberculosis.

Publication Date: 2012 Feb 17 PMID: 22206988
Authors: Goyal, A. - Verma, P. - Anandhakrishnan, M. - Gokhale, R. S. - Sankaranarayanan, R.
Journal: J Mol Biol

Activation of fatty acids as acyl-adenylates by fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis is a variant of a classical theme that involves formation of acyl-CoA (coenzyme A) by fatty acyl-CoA ligases (FACLs). Here, we show that FAALs and FACLs possess similar structural fold and substrate specificity determinants, and the key difference is the absence of a unique insertion sequence in FACL13 structure. A systematic analysis shows a conserved hydrophobic anchorage of the insertion motif across several FAALs. Strikingly, mutagenesis of two phenylalanine residues, which are part of the anchorage, to alanine converts FAAL32 to FACL32. This insertion-based in silico analysis suggests the presence of FAAL homologues in several other non-mycobacterial genomes including eukaryotes. The work presented here establishes an elegant mechanism wherein an insertion sequence drives the functional divergence of FAALs from canonical FACLs.

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Molecular Structure and Peptidoglycan Recognition of Mycobacterium tuberculosis ArfA (Rv0899).

Publication Date: 2012 Feb 17 PMID: 22206986
Authors: Yao, Y. - Barghava, N. - Kim, J. - Niederweis, M. - Marassi, F. M.
Journal: J Mol Biol

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.

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NMR Characterization of Monomeric and Oligomeric Conformations of Human Calcitonin and Its Interaction with EGCG.

Publication Date: 2012 Feb 10 PMID: 22200484
Authors: Huang, R. - Vivekanandan, S. - Brender, J. R. - Abe, Y. - Naito, A. - Ramamoorthy, A.
Journal: J Mol Biol

Calcitonin is a 32-residue peptide hormone known for its hypocalcemic effect and its inhibition of bone resorption. While calcitonin has been used in therapy for osteoporosis and Paget's disease for decades, human calcitonin (hCT) forms fibrils in aqueous solution that limit its therapeutic application. The molecular mechanism of fiber formation by calcitonin is not well understood. Here, high-resolution structures of hCT at concentrations of 0.3 mM and 1 mM have been investigated using NMR spectroscopy. Comparing the structures of hCT at different concentrations, we discovered that the peptide undergoes a conformational transition from an extended to a beta-hairpin structure in the process of molecular association. This conformational transition locates the aromatic side chains of Tyr12 and Phe16 in a favorable way for intermolecular pi-pi stacking, which is proposed to be a crucial interaction for peptide association and fibrillation. One-dimensional (1)H NMR experiments confirm that oligomerization of hCT accompanies the conformational transition at 1 mM concentration. The effect of the polyphenol epigallocatechin 3-gallate (EGCG) on hCT fibrillation was also investigated by NMR and electron microscopy, which show that EGCG efficiently inhibits fibril formation of hCT by preventing the initial association of hCT before fiber formation. The NMR experiments also indicate that the interaction between aromatic rings of EGCG and the aromatic side chains of the peptide may play an important role in inhibiting fibril formation of hCT.

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Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold.

Publication Date: 2012 Jan 13 PMID: 22198408
Authors: Koide, A. - Wojcik, J. - Gilbreth, R. N. - Hoey, R. J. - Koide, S.
Journal: J Mol Biol

The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a beta-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed "monobodies") to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.

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(phi,psi)(2) Motifs: A Purely Conformation-Based Fine-Grained Enumeration of Protein Parts at the Two-Residue Level.

Publication Date: 2012 Feb 10 PMID: 22198294
Authors: Hollingsworth, S. A. - Lewis, M. C. - Berkholz, D. S. - Wong, W. K. - Karplus, P. A.
Journal: J Mol Biol

A deep understanding of protein structure benefits from the use of a variety of classification strategies that enhance our ability to effectively describe local patterns of conformation. Here, we use a clustering algorithm to analyze 76,533 all-trans segments from protein structures solved at 1.2 A resolution or better to create a purely phi,psi-based comprehensive empirical categorization of common conformations adopted by two adjacent phi,psi pairs (i.e., (phi,psi)(2) motifs). The clustering algorithm works in an origin-shifted four-dimensional space based on the two phi,psi pairs to yield a parameter-dependent list of (phi,psi)(2) motifs, in order of their prominence. The results are remarkably distinct from and complementary to the standard hydrogen-bond-centered view of secondary structure. New insights include an unprecedented level of precision in describing the phi,psi angles of both previously known and novel motifs, ordering of these motifs by their population density, a data-driven recommendation that the standard C(alpha(i))...C(alpha(i+3))<7 A criteria for defining turns be changed to 6.5 A, identification of beta-strand and turn capping motifs, and identification of conformational capping by residues in polypeptide II conformation. We further document that the conformational preferences of a residue are substantially influenced by the conformation of its neighbors, and we suggest that accounting for these dependencies will improve protein modeling accuracy. Although the CUEVAS-4D(r(10)small je, Ukrainian(14)) 'parts list' presented here is only an initial exploration of the complex (phi,psi)(2) landscape of proteins, it shows that there is value to be had from this approach, and it opens the door to more in-depth characterizations at the (phi,psi)(2) level and at higher dimensions.

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Modulating protein-protein interactions with small molecules: the importance of binding hotspots.

Publication Date: 2012 Jan 13 PMID: 22198293
Authors: Thangudu, R. R. - Bryant, S. H. - Panchenko, A. R. - Madej, T.
Journal: J Mol Biol

The modulation of protein-protein interactions (PPIs) by small drug-like molecules is a relatively new area of research and has opened up new opportunities in drug discovery. However, the progress made in this area is limited to a handful of known cases of small molecules that target specific diseases. With the increasing availability of protein structure complexes, it is highly important to devise strategies exploiting homologous structure space on a large scale for discovering putative PPIs that could be attractive drug targets. Here, we propose a scheme that allows performing large-scale screening of all protein complexes and finding putative small-molecule and/or peptide binding sites overlapping with protein-protein binding sites (so-called "multibinding sites"). We find more than 600 nonredundant proteins from 60 protein families with multibinding sites. Moreover, we show that the multibinding sites are mostly observed in transient complexes, largely overlap with the binding hotspots and are more evolutionarily conserved than other interface sites. We investigate possible mechanisms of how small molecules may modulate protein-protein binding and discuss examples of new candidates for drug design.

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Dissection of the Network of Interactions That Links RNA Processing with Glycolysis in the Bacillus subtilis Degradosome.

Publication Date: 2012 Feb 10 PMID: 22198292
Authors: Newman, J. A. - Hewitt, L. - Rodrigues, C. - Solovyova, A. S. - Harwood, C. R. - Lewis, R. J.
Journal: J Mol Biol

The RNA degradosome is a multiprotein macromolecular complex that is involved in the degradation of messenger RNA in bacteria. The composition of this complex has been found to display a high degree of evolutionary divergence, which may reflect the adaptation of species to different environments. Recently, a degradosome-like complex identified in Bacillus subtilis was found to be distinct from those found in proteobacteria, the degradosomes of which are assembled around the unstructured C-terminus of ribonuclease E, a protein not present in B. subtilis. In this report, we have investigated in vitro the binary interactions between degradosome components and have characterized interactions between glycolytic enzymes, RNA-degrading enzymes, and those that appear to link these two cellular processes. The crystal structures of the glycolytic enzymes phosphofructokinase and enolase are presented and discussed in relation to their roles in the mediation of complex protein assemblies. Taken together, these data provide valuable insights into the structure and dynamics of the RNA degradosome, a fascinating and complex macromolecular assembly that links RNA degradation with central carbon metabolism.

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The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution.

Publication Date: 2012 Feb 10 PMID: 22197379
Authors: Amar, D. - Berger, I. - Amara, N. - Tafa, G. - Meijler, M. M. - Aharoni, A.
Journal: J Mol Biol

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.

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ATP Binding and Hydrolysis-Driven Rate-Determining Events in the RFC-Catalyzed PCNA Clamp Loading Reaction.

Publication Date: 2012 Feb 17 PMID: 22197378
Authors: Sakato, M. - Zhou, Y. - Hingorani, M. M.
Journal: J Mol Biol

The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp.DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at approximately 2 s(-1)) and bind primer-template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC.ATP.PCNA(open).ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s(-1)). Dissociation of PCNA.ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.

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Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.

Publication Date: 2012 Feb 10 PMID: 22197376
Authors: Karlsson, A. J. - Lim, H. K. - Xu, H. - Rocco, M. A. - Bratkowski, M. A. - Ke, A. - Delisa, M. P.
Journal: J Mol Biol

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.

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A Central Swivel Point in the RFC Clamp Loader Controls PCNA Opening and Loading on DNA.

Publication Date: 2012 Feb 17 PMID: 22197374
Authors: Sakato, M. - O'Donnell, M. - Hingorani, M. M.
Journal: J Mol Biol

Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA(+) superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA.ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA.ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of 'arginine-finger' ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC.ATP.PCNA(open).ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.

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Basic N-Terminus of Yeast Nhp6A Regulates the Mechanism of Its DNA Flexibility Enhancement.

Publication Date: 2012 Feb 10 PMID: 22197373
Authors: Zhang, J. - McCauley, M. J. - Maher, L. J. 3rd - Williams, M. C. - Israeloff, N. E.
Journal: J Mol Biol

HMGB (high-mobility group box) proteins are members of a class of small proteins that are ubiquitous in eukaryotic cells and nonspecifically bind to DNA, inducing large-angle DNA bends, enhancing the flexibility of DNA, and likely facilitating numerous important biological interactions. To determine the nature of this behavior for different HMGB proteins, we used atomic force microscopy to quantitatively characterize the bend angle distributions of DNA complexes with human HMGB2(Box A), yeast Nhp6A, and two chimeric mutants of these proteins. While all of the HMGB proteins bend DNA to preferred angles, Nhp6A promoted the formation of higher-order oligomer structures and induced a significantly broader distribution of angles, suggesting that the mechanism of Nhp6A is like a flexible hinge more than that of HMGB2(Box A). To determine the structural origins of this behavior, we used portions of the cationic N-terminus of Nhp6A to replace corresponding HMGB2(Box A) sequences. We found that the oligomerization and broader angle distribution correlated directly with the length of the N-terminus incorporated into the HMGB2(Box A) construct. Therefore, the basic N-terminus of Nhp6A is responsible for its ability to act as a flexible hinge and to form high-order structures.

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Two Immunoglobulin Tandem Proteins with a Linking beta-Strand Reveal Unexpected Differences in Cooperativity and Folding Pathways.

Publication Date: 2012 Feb 10 PMID: 22197372
Authors: Steward, A. - Chen, Q. - Chapman, R. I. - Borgia, M. B. - Rogers, J. M. - Wojtala, A. - Wilmanns, M. - Clarke, J.
Journal: J Mol Biol

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164-A165 and A168-A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous beta-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164-A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168-A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164-A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.

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Ligand Binding and Membrane Insertion Compete with Oligomerization of the BclXL Apoptotic Repressor.

Publication Date: 2012 Feb 10 PMID: 22197371
Authors: Bhat, V. - McDonald, C. B. - Mikles, D. C. - Deegan, B. J. - Seldeen, K. L. - Bates, M. L. - Farooq, A.
Journal: J Mol Biol

B-cell lymphoma extra large (BclXL) apoptotic repressor plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a myriad of biophysical techniques, we provide evidence that ligand binding and membrane insertion compete with oligomerization of BclXL in solution. Of particular importance is the observation that such oligomerization is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa. Binding of BH3 ligands to the canonical hydrophobic groove displaces the TM domain in a competitive manner, allowing BclXL to dissociate into monomers upon hetero-association. Remarkably, spontaneous insertion of BclXL into DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-p hosphocholine) bicelles results in a dramatic conformational change such that it can no longer recognize the BH3 ligands in what has come to be known as the "hit-and-run" mechanism. Collectively, our data suggest that oligomerization of a key apoptotic repressor serves as an allosteric switch that fine-tunes its ligand binding and membrane insertion pertinent to the regulation of apoptotic machinery.

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p53 Requires an Intact C-Terminal Domain for DNA Binding and Transactivation.

Publication Date: 2012 Feb 3 PMID: 22178617
Authors: Kim, H. - Kim, K. - Choi, J. - Heo, K. - Baek, H. J. - Roeder, R. G. - An, W.
Journal: J Mol Biol

The tumor suppressor p53 plays a critical role in mediating cellular response to a wide range of environmental stresses. p53 regulates these processes mainly by acting as a short-lived DNA binding protein that stimulates transcription from numerous genes involved in cell cycle arrest, programmed cell death, and other processes. To investigate the importance of the C-terminal domain of p53, we generated a series of deletion and point mutations in this region and analyzed their effects on p53 transcription activity. Our results show that C-terminal deletion and point mutations at K320 and K382 abolish p53-mediated transcription in the context of DNA or chromatin. This defect is specific for DNA molecules because inactive mutants fail to bind a consensus p53 response element in both free DNA and nucleosomes. Chromatin immunoprecipitation assays further substantiate the importance of the p53 C-terminal domain for the targeted localization of p53 and the concomitant recruitment of p300 onto p53-responsive genes. Moreover, a synthetic peptide comprising the last 30 amino acids of p53 interacts with the N-terminal and C-terminal domains of p53 and antagonizes p53-dependent transcription. Taken together, our data reveal a functional requirement for the p53 C-terminal domain in p53 transactivation and support a working model in which the C-terminus serves as a positive regulator for N-terminal activation and central DNA binding domains.

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Short peptides act as inducers, anti-inducers and corepressors of tet repressor.

Publication Date: 2012 Feb 10 PMID: 22178480
Authors: Goeke, D. - Kaspar, D. - Stoeckle, C. - Grubmuller, S. - Berens, C. - Klotzsche, M. - Hillen, W.
Journal: J Mol Biol

Protein allostery plays a pivotal role in many regulatory processes. Prominent examples are cell-surface receptors, which allosterically transmit ligand-generated signals to their cytoplasmic domains, or bacterial transcription factors, which alternate between a free conformation and a DNA-bound conformation in response to binding an effector molecule. The bacterial transcription factor Tet repressor (TetR) belongs to the latter category and is regarded as highly adapted to tetracyclines (tc's) as effectors. However, peptides isolated in this study were able to trigger distinct allosteric behavior including induction, anti-induction and corepression. Binding of the peptides' C-terminal residues consistently occurs within the tc-binding pocket of TetR. However, an extensive analysis of TetR mutants revealed that inducing and anti-inducing peptides utilize different parts of the binding pocket to elicit their respective regulatory responses. This study demonstrates that even for transcription factors evolved for high effector specificity, alternative molecular structures can exert similar and even novel effects, provided that sufficient chemical diversity and molecular flexibility, as found in peptide libraries, is accompanied by an efficient in vivo selection system. The high number of bioactive peptides and their extensive sequence diversity suggests that switching from small-molecule-controlled transcription regulation to a signal transduction network might be rather easily accomplished. These findings will strongly affect protein-mediated regulation of gene expression.

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An Exclusive alpha/beta Code Directs Allostery in TetR-Peptide Complexes.

Publication Date: 2012 Feb 10 PMID: 22178479
Authors: Sevvana, M. - Goetz, C. - Goeke, D. - Wimmer, C. - Berens, C. - Hillen, W. - Muller, Y. A.
Journal: J Mol Biol

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended beta-like conformation, two anti-inducing peptides form an alpha-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function.

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Inhibiting the Nucleation of Amyloid Structure in a Huntingtin Fragment by Targeting alpha-Helix-Rich Oligomeric Intermediates.

Publication Date: 2012 Feb 3 PMID: 22178478
Authors: Mishra, R. - Jayaraman, M. - Roland, B. P. - Landrum, E. - Fullam, T. - Kodali, R. - Thakur, A. K. - Arduini, I. - Wetzel, R.
Journal: J Mol Biol

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the alpha-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with d-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic alpha-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

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A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili.

Publication Date: 2012 Feb 3 PMID: 22178477
Authors: Andersson, M. - Bjornham, O. - Svantesson, M. - Badahdah, A. - Uhlin, B. E. - Bullitt, E.
Journal: J Mol Biol

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.

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Structural Insights into the Allosteric Effects of 4EBP1 on the Eukaryotic Translation Initiation Factor eIF4E.

Publication Date: 2012 Feb 3 PMID: 22178476
Authors: Siddiqui, N. - Tempel, W. - Nedyalkova, L. - Volpon, L. - Wernimont, A. K. - Osborne, M. J. - Park, H. W. - Borden, K. L.
Journal: J Mol Biol

The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5'cap) on the 5'-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5'cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5'cap.

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The formation of a potential spring in the ribosome.

Publication Date: 2012 Feb 3 PMID: 22178475
Authors: Hedrick, E. G. - Tanner, D. R. - Baig, A. - Hill, W. E.
Journal: J Mol Biol

Time-dependent chemical modification and cleavage results have provided intriguing insights into structural changes that occur in the distal loop of helix 11 in 16S ribosomal RNA (rRNA). Located distant from the decoding region, between proteins S17 and S20, the results of this study suggest that this region of rRNA may act as a buffer or a spring between these two proteins during protein biosynthesis. During the assembly process, protein S17 apparently produces the major structural deformations in this region, causing it to be folded in a spring-like structure. Base C264 in this region shows erratic behavior during assembly and also shows time-dependent enhancement when elongation factor G with GTP is added to 70S ribosomes. Evidence is presented to suggest that this region of rRNA may be used to allow relative motion to occur between proteins S17 and S20 during translocation.

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Slow Amyloid Nucleation via alpha-Helix-Rich Oligomeric Intermediates in Short Polyglutamine-Containing Huntingtin Fragments.

Publication Date: 2012 Feb 3 PMID: 22178474
Authors: Jayaraman, M. - Kodali, R. - Sahoo, B. - Thakur, A. K. - Mayasundari, A. - Mishra, R. - Peterson, C. B. - Wetzel, R.
Journal: J Mol Biol

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form alpha-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in beta-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and alpha-helical tetramer. Higher order, alpha-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ beta-sheet aggregates. These final amyloid-like aggregates not only feature the expected high beta-sheet content but also retain an element of solvent-exposed alpha-helix. The alpha-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of alpha-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.

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Functional Defect and Restoration of Temperature-Sensitive Mutants of FlhA, a Subunit of the Flagellar Protein Export Apparatus.

Publication Date: 2012 Feb 3 PMID: 22178139
Authors: Shimada, M. - Saijo-Hamano, Y. - Furukawa, Y. - Minamino, T. - Imada, K. - Namba, K.
Journal: J Mol Biol

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhA(C)) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone-substrate complexes. FlhA(C) is composed of a flexible linker region and four compact domains (A(C)D1-A(C)D4). At 42 degrees C, a temperature-sensitive (TS) G368C mutation in FlhA(C) blocks the export process after the FliH-FliI-FliJ-substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhA(C)(G368C), and its pseudorevertant variants FlhA(C)(G368C/L359F), FlhA(C)(G368C/G364R), FlhA(C)(G368C/R370S), and FlhA(C)(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhA(C) occurs in a single step, FlhA(C)(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhA(C)(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of A(C)D2 and A(C)D1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhA(C)(G368C) and that the loss of function is caused by denaturation of A(C)D2. We suggest that A(C)D2 is directly involved in the translocation of export substrates.

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Protein Kinase Domain of CTR1 from Arabidopsis thaliana Promotes Ethylene Receptor Cross Talk.

Publication Date: 2012 Jan 27 PMID: 22155294
Authors: Mayerhofer, H. - Panneerselvam, S. - Mueller-Dieckmann, J.
Journal: J Mol Biol

Ethylene controls many aspects of plant growth and development. Signaling by the gaseous phytohormone is initiated by disulfide-linked membrane-bound receptors, and the formation of heteromeric receptor clusters contributes to the broad range of ethylene responsiveness. In Arabidopsis thaliana, the TCS-like ethylene receptors interact with the cytosolic serine/threonine kinase constitutive triple response 1 (CTR1), a proposed mitogen-activated protein kinase kinase kinase. In the absence of the hormone, the receptor and therefore CTR1 are active. Hence, ethylene acts as an inverse agonist of its signaling pathway. The three-dimensional structures of the active, triphosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine illustrate the conformational rearrangements that form the basis of activity regulation. Additionally, in analytical ultracentrifugation experiments, active kinase domains form back-to-back dimers, while inactive and activation loop variants are monomers. Together with a front-to-front activation interface, the active protein kinase dimers thereby engage in interactions that promote CTR1-mediated cross talk between ethylene receptor clusters. This model provides a structural foundation for the observed high sensitivity of plants to ethylene.

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The Receptor-CheW Binding Interface in Bacterial Chemotaxis.

Publication Date: 2012 Jan 27 PMID: 22155081
Authors: Vu, A. - Wang, X. - Zhou, H. - Dahlquist, F. W.
Journal: J Mol Biol

The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the beta-strand 8 of domain 1 and the beta-strands 1 and 3 of domain 2. The receptor-CheW complex shares a similar binding interface to that found in the "trimer-of-dimers" oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor-CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW.

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Unwinding Initiation by the Viral RNA Helicase NPH-II.

Publication Date: 2012 Feb 3 PMID: 22155080
Authors: Fairman-Williams, M. E. - Jankowsky, E.
Journal: J Mol Biol

Viral RNA helicases of the NS3/NPH-II group unwind RNA duplexes by processive, directional translocation on one of the duplex strands. The translocation is preceded by a poorly understood unwinding initiation phase. For NPH-II from vaccinia virus, unwinding initiation is rate limiting for the overall unwinding reaction. To develop a mechanistic understanding of the unwinding initiation, we studied kinetic and thermodynamic aspects of this reaction phase for NPH-II in vitro, using biochemical and single molecule fluorescence approaches. Our data show that NPH-II functions as a monomer and that different stages of the ATP hydrolysis cycle dictate distinct binding preferences of NPH-II for duplex versus single-stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single conformation but rather at least two distinct conformations in each of the analyzed stages of ATP hydrolysis. These conformations interconvert with rate constants that depend on the stage of the ATP hydrolysis cycle. Our data establish a basic mechanistic framework for unwinding initiation by NPH-II and suggest that the various stages of the ATP hydrolysis cycle do not induce single, stage-specific conformations in the NPH-II-RNA complex but primarily control transitions between multiple states.

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Effects of pathogenic proline mutations on Myosin assembly.

Publication Date: 2012 Feb 3 PMID: 22155079
Authors: Buvoli, M. - Buvoli, A. - Leinwand, L. A.
Journal: J Mol Biol

Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the beta-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the alpha-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease.

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Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily.

Publication Date: 2012 Jan 27 PMID: 22155078
Authors: Gajewski, S. - Comeaux, E. Q. - Jafari, N. - Bharatham, N. - Bashford, D. - White, S. W. - van Waardenburg, R. C.
Journal: J Mol Biol

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines-one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK(a) of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.

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Inhibition of Pseudomonas aeruginosa Virulence: Characterization of the AprA-AprI Interface and Species Selectivity.

Publication Date: 2012 Jan 20 PMID: 22154939
Authors: Bardoel, B. W. - van Kessel, K. P. - van Strijp, J. A. - Milder, F. J.
Journal: J Mol Biol

Pseudomonas aeruginosa secretes the virulence factor alkaline protease (AprA) to enhance its survival. AprA cleaves one of the key microbial recognition molecules, monomeric flagellin, and thereby diminishes Toll-like receptor 5 activation. In addition, AprA degrades host proteins such as complement proteins and cytokines. P. aeruginosa encodes a highly potent inhibitor of alkaline protease (AprI) that is solely located in the periplasm where it is presumed to protect periplasmic proteins against secreted AprA. We set out to study the enzyme-inhibitor interactions in more detail in order to provide a basis for future drug development. Structural and mutational studies reveal that the conserved N-terminal residues of AprI occupy the protease active site and are essential for inhibitory activity. We constructed peptides mimicking the N-terminus of AprI; however, these were incapable of inhibiting AprA-mediated flagellin cleavage. Furthermore, we expressed and purified AprI of P. aeruginosa and the homologous (37% sequence identity) AprI of Pseudomonas syringae, which remarkably show species specificity for their cognate protease. Exchange of the first five N-terminal residues between AprI of P. syringae and P. aeruginosa did not affect the observed specificity, whereas exchange of only six residues located at the AprI surface that contacts the protease did abolish specificity. These findings are elementary steps toward the design of molecules derived from the natural inhibitor of the virulence factor AprA and their use in therapeutic applications in Pseudomonas and other Gram-negative infections.

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A reverse binding motif that contributes to specific protease inhibition by antibodies.

Publication Date: 2012 Jan 27 PMID: 22154938
Authors: Schneider, E. L. - Lee, M. S. - Baharuddin, A. - Goetz, D. H. - Farady, C. J. - Ward, M. - Wang, C. I. - Craik, C. S.
Journal: J Mol Biol

The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naive B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of approximately 200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 A and 1.5 A, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications.

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Human DNA Polymerase eta Is Pre-Aligned for dNTP Binding and Catalysis.

Publication Date: 2012 Jan 27 PMID: 22154937
Authors: Ummat, A. - Silverstein, T. D. - Jain, R. - Buku, A. - Johnson, R. E. - Prakash, L. - Prakash, S. - Aggarwal, A. K.
Journal: J Mol Biol

Pre-steady-state kinetic studies on Y-family DNA polymerase eta (Poleta) have suggested that the polymerase undergoes a rate-limiting conformational change step before the phosphoryl transfer of the incoming nucleotide to the primer terminus. However, the nature of this rate-limiting conformational change step has been unclear, due in part to the lack of structural information on the Poleta binary complex. We present here for the first time a crystal structure of human Poleta (hPoleta) in binary complex with its DNA substrate. We show that the hPoleta domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration and that the active site is devoid of metals in the absence of dNTP. Together, these observations lead us to suggest that the rate-limiting conformational change step in the Poleta replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site.

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Solution Structure of the HIV-1 Exon Splicing Silencer 3.

Publication Date: 2012 Jan 27 PMID: 22154809
Authors: Levengood, J. D. - Rollins, C. - Mishler, C. H. - Johnson, C. A. - Miner, G. - Rajan, P. - Znosko, B. M. - Tolbert, B. S.
Journal: J Mol Biol

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.8+/-1.1 nM) complex with ESS3 via site-specific interactions with the loop.

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Three RNA Recognition Motifs Participate in RNA Recognition and Structural Organization by the Pro-Apoptotic Factor TIA-1.

Publication Date: 2012 Jan 27 PMID: 22154808
Authors: Bauer, W. J. - Heath, J. - Jenkins, J. L. - Kielkopf, C. L.
Journal: J Mol Biol

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.

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Dynamic Cross-Talk among Remote Binding Sites: The Molecular Basis for Unusual Synergistic Allostery.

Publication Date: 2012 Jan 27 PMID: 22154807
Authors: Jiao, W. - Hutton, R. D. - Cross, P. J. - Jameson, G. B. - Parker, E. J.
Journal: J Mol Biol

Allosteric regulation of protein function is critical for metabolic control. Binding of allosteric effectors elicits a functional change in a remote ligand binding site on a protein by altering the equilibrium between different forms in the protein ensemble. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids Trp, Phe, and Tyr. Feedback regulation by the aromatic amino acids is important for controlling the cellular levels of the aromatic amino acids, and many organisms have two or more DAH7PS isozymes that show differing sensitivities to aromatic compounds. Mycobacterium tuberculosis expresses a single DAH7PS that is insensitive to the presence of a single amino acid yet shows extraordinary synergistic inhibition by combinations of the pathway end products Trp and Phe. The Trp+Phe-bound structure for M. tuberculosis DAH7PS, showing two separate binding sites occupied by Trp and Phe for each monomer of the tetrameric protein, was obtained by cocrystallization. Comparison of this structure with the ligand-free M. tuberculosis DAH7PS demonstrates that there is no significant change in conformation upon ligand binding, suggesting that contributions from altered dynamic properties of the enzyme may account for the allosteric inhibition. Isothermal titration calorimetry experiments demonstrate that the inhibitor binding sites are in direct communication. Molecular dynamics simulations reveal different changes in dynamic fluctuations upon single ligand binding compared to dual ligand binding. These changes account for the cross-talk between inhibitor binding sites and the active site, simultaneously potentiating both dual ligand binding and diminution of catalytic function.

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CECR2 Is Involved in Spermatogenesis and Forms a Complex with SNF2H in the Testis.

Publication Date: 2012 Feb 3 PMID: 22154806
Authors: Thompson, P. J. - Norton, K. A. - Niri, F. H. - Dawe, C. E. - McDermid, H. E.
Journal: J Mol Biol

The regulation of nucleosome positioning and composition by ATP-dependent chromatin remodeling enzymes and their associated binding partners plays important biological roles in mammals. CECR2 is a binding partner to the ISWI (imitation switch) ATPase SNF2L/SMARCA1 and is involved in neural tube closure and inner ear development; however, its functions in adult tissues have not been examined. Here, we report that CECR2 contributes to spermatogenesis and forms a complex that includes the other ISWI ATPase SNF2H/SMARCA5 in the testis. Cecr2 mutant males non-penetrant for neural tube defects sired smaller litters than wild-type males. Strikingly, while we found that Cecr2 mutants have normal seminiferous epithelium morphology, sperm count, motility, and morphology, the mutant spermatozoa were compromised in their ability to fertilize oocytes. Investigation of CECR2/ISWI complexes in the testis showed that SNF2H interacted with CECR2, and this interaction was also observed in embryonic stem cells, suggesting that CECR2 may interact with SNF2H or SNF2L depending on the cell type. Finally, we found that Cecr2 mutants exhibit misregulation of the homeobox transcription factor Dlx5 in the testis, suggesting that CECR2 complexes may regulate gene expression during spermatogenesis. Taken together, our results demonstrate a novel role of CECR2-containing complexes in spermatogenesis and show that CECR2 interacts predominantly with SNF2H instead of SNF2L in the testis.

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A conserved glu-arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold.

Publication Date: 2012 Jan 27 PMID: 22138346
Authors: Yang, J. - Wu, J. - Steichen, J. M. - Kornev, A. P. - Deal, M. S. - Li, S. - Sankaran, B. - Woods, V. L. Jr - Taylor, S. S.
Journal: J Mol Biol

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises alphaG-alphaH-alphaI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.

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Crystal Structures of Two Solute Receptors for l-Cystine and l-Cysteine, Respectively, of the Human Pathogen Neisseria gonorrhoeae.

Publication Date: 2012 Jan 20 PMID: 22138345
Authors: Bulut, H. - Moniot, S. - Licht, A. - Scheffel, F. - Gathmann, S. - Saenger, W. - Schneider, E.
Journal: J Mol Biol

ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free "open" conformation and in a "closed" conformation when co-crystallized with l-cystine. Our data provide the first structural information of an l-cystine ABC transporter. Dissociation constants of 21 and 33 nM for l-cystine and l-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, l-cystathionine and l-djenkolic acid are weak binders, while no binding was detectable for S-methyl-l-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for l-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified l-cysteine. The protein binds l-cysteine with a K(d) of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since l-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine.

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Salt Bridges Regulate Both Dimer Formation and Monomeric Flexibility in HdeB and May Have a Role in Periplasmic Chaperone Function.

Publication Date: 2012 Jan 20 PMID: 22138344
Authors: Wang, W. - Rasmussen, T. - Harding, A. J. - Booth, N. A. - Booth, I. R. - Naismith, J. H.
Journal: J Mol Biol

Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.

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Structural Studies of Intermediates along the Cyclization Pathway of Aplysia ADP-Ribosyl Cyclase.

Publication Date: 2012 Jan 20 PMID: 22138343
Authors: Kotaka, M. - Graeff, R. - Chen, Z. - Zhang, L. H. - Lee, H. C. - Hao, Q.
Journal: J Mol Biol

Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca(2+) stores and activate Ca(2+) influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD(+) into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase.

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The H2A-H2B Dimeric Kinetic Intermediate Is Stabilized by Widespread Hydrophobic Burial with Few Fully Native Interactions.

Publication Date: 2012 Jan 20 PMID: 22137897
Authors: Guyett, P. J. - Gloss, L. M.
Journal: J Mol Biol

The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within approximately 5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I(2) and I(2)(), the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I(2) ensemble folds to the native heterodimer, N(2), through an observable, first-order kinetic phase. To determine the regions of structure in the I(2) ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I(2), the transition state and N(2); however, only residues in the hydrophobic core of the dimer interface perturbed the I(2)() population. Destabilization of I(2)() correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Phi values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I(2) and N(2), but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I(2). These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I(2), but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N(2), more native-like interactions are developed and nonnative interactions are rearranged.

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Facilitated assembly of the preinitiation complex by separated tail and head/middle modules of the mediator.

Publication Date: 2012 Jan 20 PMID: 22137896
Authors: Galdieri, L. - Desai, P. - Vancura, A.
Journal: J Mol Biol

Mediator is a general coactivator of RNA polymerase II (RNA pol II) bridging enhancer-bound transcriptional factors with RNA pol II. Mediator is organized in three distinct subcomplexes: head, middle, and tail modules. The head and middle modules interact with RNA pol II, and the tail module interacts with transcriptional activators. Deletion of one of the tail subunits SIN4 results in derepression of a subset of genes, including FLR1, by a largely unknown mechanism. Here we show that derepression of FLR1 transcription in sin4Delta cells occurs by enhanced recruitment of the mediator as well as Swi/Snf and SAGA complexes. The tail and head/middle modules of the mediator behave as separate complexes at the induced FLR1 promoter. While the tail module remains anchored to the promoter, the head/middle modules are also found in the coding region. The separation of the tail and head/middle modules in sin4Delta cells is also supported by the altered stoichiometry of the tail and head/middle modules at several tested promoters. Deletion of another subunit of the tail module MED2 in sin4Delta cells results in significantly decreased transcription of FLR1, pointing to the importance of the integrity of the separated tail module in derepression. All tested genes exhibited increased recruitment of the tail domain; however, only genes with increased occupancy of the head/middle modules also displayed increased transcription. The separated tail module thus represents a promiscuous transcriptional factor that binds to many different promoters and is necessary for derepression of FLR1 in sin4Delta cells.

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Crystal Structures of Mutant IspH Proteins Reveal a Rotation of the Substrate's Hydroxymethyl Group during Catalysis.

Publication Date: 2012 Feb 10 PMID: 22137895
Authors: Span, I. - Grawert, T. - Bacher, A. - Eisenreich, W. - Groll, M.
Journal: J Mol Biol

Isoprenoids derive from two universal precursors, isopentenyl diphosphate and dimethylallyl diphosphate, which in most human pathogens are synthesized in the deoxyxylulose phosphate pathway. The last step of this pathway is the conversion of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate catalyzed by the iron-sulfur protein IspH. The crystal structures reported here of the IspH mutant proteins T167C, E126D and E126Q reveal an alternative substrate conformation compared to the wild-type structure. Thus, the previously observed alkoxide complex decomposes, and the substrate's hydroxymethyl group rotates to interact with Glu126. The carboxyl group of Glu126 then donates a proton to the hydroxyl group to enable water elimination. The structural and functional studies provide further knowledge of the IspH reaction mechanism, which opens up new routes to inhibitor design against malaria and tuberculosis.

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Crystal Structure of Human Mitochondrial PheRS Complexed with tRNA(Phe) in the Active "Open" State.

Publication Date: 2012 Jan 20 PMID: 22137894
Authors: Klipcan, L. - Moor, N. - Finarov, I. - Kessler, N. - Sukhanova, M. - Safro, M. G.
Journal: J Mol Biol

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 A. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1-C72 base pair, and the "discriminator" base A73, proposed to contribute to tRNA(Phe) identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS-tRNA(Phe) provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the "closed" to the productive "open" state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions.

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The Structure and Enzymatic Properties of a Novel RNase II Family Enzyme from Deinococcus radiodurans.

Publication Date: 2012 Jan 20 PMID: 22133431
Authors: Schmier, B. J. - Seetharaman, J. - Deutscher, M. P. - Hunt, J. F. - Malhotra, A.
Journal: J Mol Biol

Exoribonucleases are vital in nearly all aspects of RNA metabolism, including RNA maturation, end-turnover, and degradation. RNase II and RNase R are paralogous members of the RNR superfamily of nonspecific, 3'-->5', processive exoribonucleases. In Escherichia coli, RNase II plays a primary role in mRNA decay and has a preference for unstructured RNA. RNase R, in contrast, is capable of digesting structured RNA and plays a role in the degradation of both mRNA and stable RNA. Deinococcus radiodurans, a radiation-resistant bacterium, contains two RNR family members. The shorter of these, DrR63, includes a sequence signature typical of RNase R, but we show here that this enzyme is an RNase II-type exonuclease and cannot degrade structured RNA. We also report the crystal structure of this protein, now termed DrII. The DrII structure reveals a truncated RNA binding region in which the N-terminal cold shock domains, typical of most RNR family nucleases, are replaced by an unusual winged helix-turn-helix domain, where the "wing" is contributed by the C-terminal S1 domain. Consistent with its truncated RNA binding region, DrII is able to remove 3' overhangs from RNA molecules closer to duplexes than do other RNase II-type enzymes. DrII also displays distinct sensitivity to pyrimidine-rich regions of single-stranded RNA and is able to process tRNA precursors with adenosine-rich 3' extensions in vitro. These data indicate that DrII is the RNase II of D. radiodurans and that its structure and catalytic properties are distinct from those of other related enzymes.

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The Structure of the NXF2/NXT1 Heterodimeric Complex Reveals the Combined Specificity and Versatility of the NTF2-Like Fold.

Publication Date: 2012 Jan 27 PMID: 22123199
Authors: Kerkow, D. E. - Carmel, A. B. - Menichelli, E. - Ambrus, G. - Hills, R. D. Jr - Gerace, L. - Williamson, J. R.
Journal: J Mol Biol

NXF1-like members of the NXF (nuclear export factor) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate-specific and tissue-specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NXT (NTF2-related export) protein is central to NXF family function; however, given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remain unclear. Here, we report the structural and functional analyses of the Caenorhabditis elegans NXF variant ceNXF2 bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity, including a probable site for phosphoregulation, have been identified. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for the colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.

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A transporter converted into a sensor, a phototaxis signaling mutant of bacteriorhodopsin at 3.0 A.

Publication Date: 2012 Jan 20 PMID: 22123198
Authors: Spudich, E. N. - Ozorowski, G. - Schow, E. V. - Tobias, D. J. - Spudich, J. L. - Luecke, H.
Journal: J Mol Biol

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 A resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C(14) group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174-Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.

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Dynamical characterization of two differentially disease associated MHC class I proteins in complex with viral and self-peptides.

Publication Date: 2012 Jan 13 PMID: 22119720
Authors: Narzi, D. - Becker, C. M. - Fiorillo, M. T. - Uchanska-Ziegler, B. - Ziegler, A. - Bockmann, R. A.
Journal: J Mol Biol

Major histocompatibility complex (MHC) class I proteins are expressed on the cell surface where they present foreign and self-peptides to effector cells of the immune system. While an understanding of the structural prerequisites for antigen presentation has already been achieved, insight into subtype- or peptide-dependent dynamical characteristics of a peptide-MHC antigen is so far largely obscure. We approached this problem by employing 400-ns molecular dynamics simulations with two human MHC class I subtypes as model systems: the ankylosing spondylitis-associated HLA-B *27:05 and the non-ankylosing spondylitis-associated HLA-B *27:09. Both proteins differ only by a micropolymorphism at the floor of the peptide binding groove (Asp116His). A viral (pLMP2) and three self-peptides (pVIPR, pGR, and TIS) were evaluated. The stability of the binding grooves was found to be both subtype dependent and peptide dependent. A detachment from the C- and/or N-terminal pockets was observed for all peptides except TIS, resulting in a stabilization of the alpha1-helix in both TIS-displaying subtypes. Estimates of the entropy associated with the bound peptides showed an increased entropy for pLMP2 presented by B *27:05 as compared to B *27:09, in contrast to the self-peptides. Additionally, the flexibility of the alpha1-helix that is probably important for receptor binding to the B27:peptide epitope is significantly enhanced for B *27:05. These in silico results show that the dynamic properties of peptide-MHC complexes are affected both by the bound peptide and by micropolymorphisms of the heavy chain. Our findings suggest a role for the conformational flexibility of MHC class I molecules in the context of recognition by receptors on effector cells.

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Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs.

Publication Date: 2012 Jan 20 PMID: 22119487
Authors: Naik, A. K. - Raghavan, S. C.
Journal: J Mol Biol

During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease.

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Amyloid Fibrils Formed by the Programmed Cell Death Regulator Bcl-xL.

Publication Date: 2012 Jan 20 PMID: 22119486
Authors: Chenal, A. - Vendrely, C. - Vitrac, H. - Karst, J. C. - Gonneaud, A. - Blanchet, C. E. - Pichard, S. - Garcia, E. - Salin, B. - Catty, P. - Gillet, D. - Hussy, N. - Marquette, C. - Almunia, C. - Forge, V.
Journal: J Mol Biol

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular beta-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.

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Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis.

Publication Date: 2012 Jan 13 PMID: 22115775
Authors: Levdikov, V. M. - Blagova, E. V. - Rawlings, A. E. - Jameson, K. - Tunaley, J. - Hart, D. J. - Barak, I. - Wilkinson, A. J.
Journal: J Mol Biol

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor sigma(F) from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 A spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded beta-sheets are packed against one another and flanked by alpha-helices in an alphabetabetaalpha arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling.

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Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics.

Publication Date: 2012 Jan 13 PMID: 22115774
Authors: Kleckner, I. R. - Gollnick, P. - Foster, M. P.
Journal: J Mol Biol

The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins.

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Challenging Packaging Limits and Infectivity of Phage lambda.

Publication Date: 2012 Jan 13 PMID: 22108169
Authors: Nurmemmedov, E. - Castelnovo, M. - Medina, E. - Catalano, C. E. - Evilevitch, A.
Journal: J Mol Biol

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage lambda and still yield infectious virions and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage lambda from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage lambda is able to produce infectious particles with up to 110% of the wild-type lambda-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length and was a factor of 10(3) lower for the 110% lambda-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the wild-type length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these findings are discussed.

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Structural Analysis of Chi1 Chitinase from Yen-Tc: The Multisubunit Insecticidal ABC Toxin Complex of Yersinia entomophaga.

Publication Date: 2012 Jan 13 PMID: 22108167
Authors: Busby, J. N. - Landsberg, M. J. - Simpson, R. M. - Jones, S. A. - Hankamer, B. - Hurst, M. R. - Lott, J. S.
Journal: J Mol Biol

Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 A resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex.

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Structural Studies of Mycobacterium tuberculosis Rv0899 Reveal a Monomeric Membrane-Anchoring Protein with Two Separate Domains.

Publication Date: 2012 Jan 13 PMID: 22108166
Authors: Li, J. - Shi, C. - Gao, Y. - Wu, K. - Shi, P. - Lai, C. - Chen, L. - Wu, F. - Tian, C.
Journal: J Mol Biol

Rv0899 from Mycobacterium tuberculosis belongs to the OmpA (outer membrane protein A) family of outer membrane proteins. It functions as a pore-forming protein; the deletion of this gene impairs the uptake of some water-soluble substances, such as serine, glucose, and glycerol. Rv0899 has also been shown to play a part in low-pH environment adaption, which may play a part in pathogenic mycobacteria overcoming the host's defense mechanisms. Based on many bacterial physiological data and recent structural studies, it was proposed that Rv0899 forms an oligomeric channel to carry out such functions. In this work, biochemical and structural data obtained from solution NMR and EPR spectroscopy indicated that Rv0899 is a monomeric membrane-anchoring protein with two separate domains, rather than an oligomeric pore. Using NMR chemical shift perturbation and isothermal calorimetric titration assays, we show that Rv0899 was able to interact with Zn(2+) ions, which may indicate a role for Rv0899 in the process of Zn(2+) acquisition.

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Altered strand transfer activity of a multiple-drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutant with a dipeptide fingers domain insertion.

Publication Date: 2012 Jan 13 PMID: 22100453
Authors: Nguyen, L. A. - Daddacha, W. - Rigby, S. - Bambara, R. A. - Kim, B.
Journal: J Mol Biol

Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (k(on)) for template binding and a lower equilibrium binding constant K(d) for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k(off) values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis.

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Structural basis for activation of calcineurin by calmodulin.

Publication Date: 2012 Jan 13 PMID: 22100452
Authors: Rumi-Masante, J. - Rusinga, F. I. - Lester, T. E. - Dunlap, T. B. - Williams, T. D. - Dunker, A. K. - Weis, D. D. - Creamer, T. P.
Journal: J Mol Biol

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an approximately 25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting alpha-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

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Crystallographic Evidence for a Domain Motion in Rat Nucleoside Triphosphate Diphosphohydrolase (NTPDase) 1.

Publication Date: 2012 Jan 13 PMID: 22100451
Authors: Zebisch, M. - Krauss, M. - Schafer, P. - Strater, N.
Journal: J Mol Biol

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a physiologically important class of membrane-bound ectonucleotidases responsible for the regulation of extracellular levels of nucleotides. CD39 or NTPDase1 is the dominant NTPDase of the vasculature. By hydrolyzing proinflammatory ATP and platelet-activating ADP to AMP, it blocks platelet aggregation and supports blood flow. Thus, great interest exists in understanding the structure and dynamics of this prototype member of the eukaryotic NTPDase family. Here, we report the crystal structure of a variant of soluble NTPDase1 lacking a putative membrane interaction loop identified between the two lobes of the catalytic domain. ATPase and ADPase activities of this variant are determined via a newly established kinetic isothermal titration calorimetry assay and compared to that of the soluble NTPDase1 variant characterized previously. Complex structures with decavanadate and heptamolybdate show that both polyoxometallates bind electrostatically to a loop that is involved in binding of the nucleobase. In addition, a comparison of the domain orientations of the four independent proteins in the crystal asymmetric unit provides the first direct experimental evidence for a domain motion of NTPDases. An interdomain rotation angle of up to 7.4 degrees affects the active site cleft between the two lobes of the protein. Comparison with a previously solved bacterial NTPDase structure indicates that the domains may undergo relative rotational movements of more than 20 degrees . Our data support the idea that the influence of transmembrane helix dynamics on activity is achieved by coupling to a domain motion.

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UHRF1 Double Tudor Domain and the Adjacent PHD Finger Act Together to Recognize K9me3-Containing Histone H3 Tail.

Publication Date: 2012 Jan 13 PMID: 22100450
Authors: Xie, S. - Jakoncic, J. - Qian, C.
Journal: J Mol Biol

Human multi-domain-containing protein UHRF1 has recently been extensively characterized as a key epigenetic regulator for maintaining DNA methylation patterns. UHRF1 SRA domain preferentially binds to hemimethylated CpG sites, and double Tudor domain has been implicated in recognizing H3K9me3 mark, but the role of the adjacent PHD finger remains unclear. Here, we report the high-resolution crystal structure of UHRF1 PHD finger in complex with N-terminal tail of histone H3. We found that the preceding zinc-Cys4 knuckle is indispensable for the PHD finger of UHRF1 to recognize the first four unmodified residues of histone H3 N-terminal tail. Quantitative binding studies indicated that UHRF1 PHD finger (including the preceding zinc-Cys4 knuckle) acts together with the adjacent double Tudor domain to specifically recognize the H3K9me3 mark. Combinatorial recognition of H3K9me3-containing histone H3 tail by UHRF1 PHD finger and double Tudor domain may play a role in establishing and maintaining histone H3K9 methylation patterns during the cell cycle.

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Crystal structure of sol I 2: a major allergen from fire ant venom.

Publication Date: 2012 Jan 27 PMID: 22100449
Authors: Borer, A. S. - Wassmann, P. - Schmidt, M. - Hoffman, D. R. - Zhou, J. J. - Wright, C. - Schirmer, T. - Markovic-Housley, Z.
Journal: J Mol Biol

Sol i 2 is a potent allergen from the venom of red imported fire ant, which contains allergens Sol i 1, Sol i 2, Sol i 3, and Sol i 4 that are known to be powerful triggers of anaphylaxis. Sol i 2 causes IgE antibody production in about one-third of individuals stung by fire ants. Baculovirus recombinant dimeric Sol i 2 was crystallized as a native and selenomethionyl-derivatized protein, and its structure has been determined by single-wavelength anomalous dispersion at 2.6 A resolution. The overall fold of each subunit consists of five helices that enclose a central hydrophobic cavity. The structure is stabilized by three intramolecular disulfide bridges and one intermolecular disulfide bridge. The nearest structural homologue is the sequence-unrelated odorant binding protein and pheromone binding protein LUSH of the fruit fly Drosophila, which may suggest a similar biological function. To test this hypothesis, we measured the reversible binding of various pheromones, plant odorants, and other ligands to Sol i 2 by the changes in N-phenyl-1-naphthylamine fluorescence emission upon binding of ligands that compete with N-phenyl-1-naphthylamine. The highest binding affinity was observed for hydrophobic ligands such as aphid alarm pheromone (E)-beta-farnesene, analogs of ant alarm pheromones, and plant volatiles decane, undecane, and beta-caryophyllene. Conceivably, Sol i 2 may play a role in capturing and/or transporting small hydrophobic ligands such as pheromones, odors, fatty acids, or short-living hydrophobic primers. Molecular surface analysis, in combination with sequence alignment, can explain the serological cross-reactivity observed between some ant species.

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A Bayesian View on Cryo-EM Structure Determination.

Publication Date: 2012 Jan 13 PMID: 22100448
Authors: Scheres, S. H.
Journal: J Mol Biol

Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures.

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Protein hot spots: the islands of stability.

Publication Date: 2012 Jan 13 PMID: 22100447
Authors: Kuttner, Y. Y. - Engel, S.
Journal: J Mol Biol

Understanding the structural basis of protein-protein interactions (PPIs) may shed light on the organization and functioning of signal transduction and metabolic networks and may assist in structure-based design of ligands (drugs) targeting protein-protein interfaces. The residues at the bimolecular interface, designated as the hot spots, contribute most of the free binding energy of PPI. To date, there is no conclusive atomistic explanation for the unique functional properties of the hot spots. We hypothesized that backbone compliance may play a role in protein-protein recognition and in the mechanism of binding of small-molecule compounds to protein surfaces. We used a steered molecular dynamics simulation to explore the compliance properties of the backbone of surface-exposed residues in several model proteins: interleukin-2, mouse double minute protein 2 and proliferating cell nuclear antigen. We demonstrated that protein surfaces exhibit distinct patterns in which highly immobile residues form defined clusters ("stability patches") alternating with areas of moderate to high mobility. These "stability patches" tend to localize in functionally important regions involved in protein-protein recognition. We propose a mechanism by which the distinct structural organization of the hot spots may contribute to their role in mediating PPI and facilitating binding of structurally diverse small-molecule compounds to protein surfaces.

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ExbD Mutants Define Initial Stages in TonB Energization.

Publication Date: 2012 Jan 13 PMID: 22100395
Authors: Ollis, A. A. - Postle, K.
Journal: J Mol Biol

Cytoplasmic membrane proteins ExbB and ExbD of the Escherichia coli TonB system couple cytoplasmic membrane protonmotive force (pmf) to TonB. TonB transmits this energy to high-affinity outer membrane active transporters. ExbD is proposed to catalyze TonB conformational changes during energy transduction. Here, the effect of ExbD mutants and changes in pmf on TonB proteinase K sensitivity in spheroplasts was examined. Spheroplasts supported the pmf-dependent formaldehyde cross-link between periplasmic domains of TonB and ExbD, indicating that they constituted a biologically relevant in vivo system to study changes in TonB proteinase K sensitivity. Three stages in TonB energization were identified. In Stage I, ExbD L123Q or TonB H20A prevented proper interaction between TonB and ExbD, rendering TonB sensitive to proteinase K. In Stage II, ExbD D25N supported conversion of TonB to a proteinase-K-resistant form, but not energization of TonB or formation of the pmf-dependent formaldehyde cross-link. Addition of protonophores had the same effect as ExbD D25N. This suggested the existence of a pmf-independent association between TonB and ExbD. TonB proceeded to Stage III when pmf was present, again becoming proteinase K sensitive, but now able to form the pmf-dependent cross-link to ExbD. Absence or presence of pmf toggled TonB between Stage II and Stage III conformations, which were also detected in wild-type cells. ExbD also underwent pmf-dependent conformational changes that were interdependent with TonB. These observations supported the hypothesis that ExbD couples TonB to the pmf, with concomitant transitions of ExbD and TonB periplasmic domains from unenergized to energized heterodimers.

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UG Repeats/TDP-43 Interactions near 5' Splice Sites Exert Unpredictable Effects on Splicing Modulation.

Publication Date: 2012 Jan 6 PMID: 22100394
Authors: Passoni, M. - De Conti, L. - Baralle, M. - Buratti, E.
Journal: J Mol Biol

TDP-43 is a nuclear protein implicated in the pathogenesis of several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration, with broad involvement in numerous stages of RNA processing ranging from transcription to translation. In diseased neurons, TDP-43 mostly aggregates in the cytoplasm, suggesting that a loss of protein function in the nucleus may play an important role in neurodegeneration. A better understanding of TDP-43 general nuclear functions is therefore an essential step to evaluate this possibility. Presently, the TDP-43 best-characterized functional property is its ability to modulate pre-mRNA splicing when binding in proximity of 3'SS acceptor sequences. In this work, using a variety of artificial and natural splicing substrates, we have investigated the effects of TDP-43 binding to UG repeats in the vicinity of 5'SS donor sequences. In general, our results show that UG repeats are not powerful splicing regulatory elements when located near to exonic 5'SS sequences. However, in cases like the BRCA1, ETF1, and RXRG genes, TDP-43 binding to natural UG-repeated sequences can act as either an activator or a suppressor of 5'SS recognition, depending on splice site strength and on the presence of additional splicing regulatory sequences. The results of this analysis suggest that a role of UG repeats/TDP-43 in 5'SS recognition may exists and may become critical in the presence of mutations that weaken the 5'SS. The general rule that can be drawn at the moment is that the importance of UG repeats near 5' splice sites should always be experimentally validated on a case-by-case basis.

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Adenine recognition is a key checkpoint in the energy release mechanism of phage t4 DNA packaging motor.

Publication Date: 2012 Jan 13 PMID: 22100308
Authors: Kondabagil, K. - Draper, B. - Rao, V. B.
Journal: J Mol Biol

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.

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Transcription Factor GATA-6 Recruits PPARalpha to Cooperatively Activate Glut4 Gene Expression.

Publication Date: 2012 Jan 6 PMID: 22100307
Authors: Yao, C. X. - Xiong, C. J. - Wang, W. P. - Yang, F. - Zhang, S. F. - Wang, T. Q. - Wang, S. L. - Yu, H. L. - Wei, Z. R. - Zang, M. X.
Journal: J Mol Biol

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear hormone receptor that regulates energy metabolism, but its precise mechanisms remain unknown. Here, we demonstrate that the PPARalpha agonist fenofibrate activated expression of the glucose transporter Glut4. Moreover, PPARalpha was associated with the Glut4 promoter through GATA sites upon fenofibrate stimulation in cardiomyocytes. This occupancy is achieved through an interaction between amino acids 1-136 of PPARalpha with amino acids 276-443 of the cardiac transcription factor GATA-6. In addition, the interaction of PPARalpha with GATA-6 activated Glut4 gene expression, improved glucose consumption, and enhanced activity of mitochondrial citrate synthase in C2C12 myoblasts; both mutants of PPARalpha (1-101 aa) and GATA-6 (227-331 aa) were unable to cooperate in Glut4 activation. Thus, GATA-6 is an important component of the transcription network required for energy metabolism mediated by PPARalpha, and these findings provide a molecular basis for understanding the role of GATA-6 proteins in muscle development and disease.

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Structure and Kinetic Stability of the p63 Tetramerization Domain.

Publication Date: 2012 Jan 20 PMID: 22100306
Authors: Natan, E. - Joerger, A. C.
Journal: J Mol Biol

The p53 family of transcription factors-comprising p53, p63 and p73-plays an important role in tumor prevention and development. Essential to their function is the formation of tetramers, allowing cooperative binding to their DNA response elements. We solved crystal structures of the human p63 tetramerization domain, showing that p63 forms a dimer of dimers with D(2) symmetry composed of highly intertwined monomers. The primary dimers are formed via an intramolecular beta-sheet and hydrophobic helix packing (H1), a hallmark of all p53 family members. Like p73, but unlike p53, p63 requires a second helix (H2) to stabilize the architecture of the tetramer. In order to investigate the impact of structural differences on tetramer stability, we measured the subunit exchange reaction of p53 family homotetramers by nanoflow electrospray mass spectrometry. There were differences in both the kinetics and the pattern of the exchange reaction, with the p53 and p63 tetramers exhibiting much faster exchange kinetics than p73. The structural similarity between p63 and p73 rationalizes previous observations that p63 and p73 form mixed tetramers, and the kinetic data reveal the dissociation of the p73 homotetramers as the rate-limiting step for heterotetramer formation. Differential stability of the tetramers may play an important role in the cross talk between different isoforms and regulation of p53, p63 and p73 function in the cell cycle.

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Decoding the Molecular Design Principles Underlying Ca(2+) Binding to betagamma-Crystallin Motifs.

Publication Date: 2012 Jan 6 PMID: 22099475
Authors: Mishra, A. - Suman, S. K. - Srivastava, S. S. - Sankaranarayanan, R. - Sharma, Y.
Journal: J Mol Biol

Numerous proteins belonging to the recently expanded betagamma-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to betagamma-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the betagamma-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a betagamma-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.

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Bound for observation.

Publication Date: 2012 Jan 6 PMID: 22094315
Authors: Bolon, D. N.
Journal: J Mol Biol

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Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.

Publication Date: 2012 Jan 6 PMID: 22094314
Authors: Barnard, T. J. - Gumbart, J. - Peterson, J. H. - Noinaj, N. - Easley, N. C. - Dautin, N. - Kuszak, A. J. - Tajkhorshid, E. - Bernstein, H. D. - Buchanan, S. K.
Journal: J Mol Biol

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded beta-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.

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POU1F1-Mediated Activation of hGH-N by Deoxyribonuclease I Hypersensitive Site II of the Human Growth Hormone Locus Control Region.

Publication Date: 2012 Jan 6 PMID: 22094313
Authors: Hunsaker, T. L. - Jefferson, H. S. - Morrison, J. K. - Franklin, A. J. - Shewchuk, B. M.
Journal: J Mol Biol

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.

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Intra-Chain 3D Segment Swapping Spawns the Evolution of New Multidomain Protein Architectures.

Publication Date: 2012 Jan 6 PMID: 22079367
Authors: Szilagyi, A. - Zhang, Y. - Zavodszky, P.
Journal: J Mol Biol

Multidomain proteins form in evolution through the concatenation of domains, but structural domains may comprise multiple segments of the chain. In this work, we demonstrate that new multidomain architectures can evolve by an apparent three-dimensional swap of segments between structurally similar domains within a single-chain monomer. By a comprehensive structural search of the current Protein Data Bank (PDB), we identified 32 well-defined segment-swapped proteins (SSPs) belonging to 18 structural families. Nearly 13% of all multidomain proteins in the PDB may have a segment-swapped evolutionary precursor as estimated by more permissive searching criteria. The formation of SSPs can be explained by two principal evolutionary mechanisms: (i) domain swapping and fusion (DSF) and (ii) circular permutation (CP). By large-scale comparative analyses using structural alignment and hidden Markov model methods, it was found that the majority of SSPs have evolved via the DSF mechanism, and a much smaller fraction, via CP. Functional analyses further revealed that segment swapping, which results in two linkers connecting the domains, may impart directed flexibility to multidomain proteins and contributes to the development of new functions. Thus, inter-domain segment swapping represents a novel general mechanism by which new protein folds and multidomain architectures arise in evolution, and SSPs have structural and functional properties that make them worth defining as a separate group.

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YhiQ Is RsmJ, the Methyltransferase Responsible for Methylation of G1516 in 16S rRNA of E. coli.

Publication Date: 2012 Jan 6 PMID: 22079366
Authors: Basturea, G. N. - Dague, D. R. - Deutscher, M. P. - Rudd, K. E.
Journal: J Mol Biol

Ten methyltransferases and one pseudouridine synthase are required for complete modification of the small ribosomal subunit in Escherichia coli. Nine methyltransferases, as well as the pseudouridine synthase, are already known. Here, we identify RsmJ, the last unknown methyltransferase required for methylation of m(2)G1516 in 16S ribosomal RNA (rRNA), as the protein encoded by yhiQ. Reverse transcription primer extension analysis reveals that rRNA extracted from a yhiQ deletion strain is not methylated at G1516. Moreover, methylation is restored upon gene complementation. Also, purified recombinant YhiQ specifically methylates 30S subunits extracted from the deletion strain. The absence of the yhiQ gene leads to a cold-sensitive phenotype. Based on these data, we propose that the yhiQ gene be renamed rsmJ.

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The Structure of Aquifex aeolicus Ribosomal Protein S8 Reveals a Unique Subdomain that Contributes to an Extremely Tight Association with 16S rRNA.

Publication Date: 2012 Jan 20 PMID: 22079365
Authors: Menichelli, E. - Edgcomb, S. P. - Recht, M. I. - Williamson, J. R.
Journal: J Mol Biol

The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA.

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Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation.

Publication Date: 2012 Jan 13 PMID: 22079364
Authors: Baumann, B. A. - Taylor, D. W. - Huang, Z. - Tama, F. - Fagnant, P. M. - Trybus, K. M. - Taylor, K. A.
Journal: J Mol Biol

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

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Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody.

Publication Date: 2012 Jan 6 PMID: 22079050
Authors: Tars, K. - Kotelovica, S. - Lipowsky, G. - Bauer, M. - Beerli, R. R. - Bachmann, M. F. - Maurer, P.
Journal: J Mol Biol

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 A resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-pi interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180 degrees . This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.

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The structure of the extracellular domain of the jumping translocation breakpoint protein reveals a variation of the midkine fold.

Publication Date: 2012 Jan 6 PMID: 22079049
Authors: Rousseau, F. - Pan, B. - Fairbrother, W. J. - Bazan, J. F. - Lingel, A.
Journal: J Mol Biol

Jumping Translocation Breakpoint (JTB) is an orphan receptor that is conserved from nematodes to humans and whose gene expression in humans is strikingly upregulated in diverse types of cancers. Translocations occur frequently at the hJTB genomic locus, leading to multiple copies of a truncated JTB gene, which potentially encodes a soluble secreted ectodomain. In addition, JTB and its orthologs likely represent a unique and ancient protein family since homologs could not be identified by direct sequence comparison. In the present study, we have determined the NMR solution structure of the N-terminal ectodomain of human JTB, showing that its fold architecture is a new variant of a three-beta-strand antiparallel beta-meander. The JTB structure has a distant relationship to the midkine/pleiotrophin fold, particularly in the conservation of distinctive disulfide bridge patterns. The structure of this newly characterized small cysteine-rich domain suggests potential involvement of JTB in interactions with proteins or extracellular matrix and may help to uncover the elusive biological functions of this protein.

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Prediction of short linear protein binding regions.

Publication Date: 2012 Jan 6 PMID: 22079048
Authors: Mooney, C. - Pollastri, G. - Shields, D. C. - Haslam, N. J.
Journal: J Mol Biol

Short linear motifs in proteins (typically 3-12 residues in length) play key roles in protein-protein interactions by frequently binding specifically to peptide binding domains within interacting proteins. Their tendency to be found in disordered segments of proteins has meant that they have often been overlooked. Here we present SLiMPred (short linear motif predictor), the first general de novo method designed to computationally predict such regions in protein primary sequences independent of experimentally defined homologs and interactors. The method applies machine learning techniques to predict new motifs based on annotated instances from the Eukaryotic Linear Motif database, as well as structural, biophysical, and biochemical features derived from the protein primary sequence. We have integrated these data sources and benchmarked the predictive accuracy of the method, and found that it performs equivalently to a predictor of protein binding regions in disordered regions, in addition to having predictive power for other classes of motif sites such as polyproline II helix motifs and short linear motifs lying in ordered regions. It will be useful in predicting peptides involved in potential protein associations and will aid in the functional characterization of proteins, especially of proteins lacking experimental information on structures and interactions. We conclude that, despite the diversity of motif sequences and structures, SLiMPred is a valuable tool for prioritizing potential interaction motifs in proteins.

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The Evolution of Cefotaximase Activity in the TEM beta-Lactamase.

Publication Date: 2012 Jan 6 PMID: 22075446
Authors: Singh, M. K. - Dominy, B. N.
Journal: J Mol Biol

The development of a molecular-level understanding of drug resistance through beta-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 beta-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants. Further analyses suggest that the changes in catalytic efficiency for mutant enzymes are correlated to structural changes within the binding site. Based on the energetic and structural analyses of the wild-type and mutant enzymes, structural rearrangement is suggested as a mechanism of evolution of drug resistance through TEM beta-lactamase. The present study not only provides molecular-level insight into the effect of four drug resistance mutations on the structure and function of the TEM beta-lactamase but also establishes a foundation for a future molecular-level analysis of complete evolutionary trajectory for this class of enzymes.

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Structural analyses of covalent enzyme-substrate analog complexes reveal strengths and limitations of de novo enzyme design.

Publication Date: 2012 Jan 20 PMID: 22075445
Authors: Wang, L. - Althoff, E. A. - Bolduc, J. - Jiang, L. - Moody, J. - Lassila, J. K. - Giger, L. - Hilvert, D. - Stoddard, B. - Baker, D.
Journal: J Mol Biol

We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 A over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and beta-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme-inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl-substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.

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Cross-monomer substrate contacts reposition the hsp90 N-terminal domain and prime the chaperone activity.

Publication Date: 2012 Jan 6 PMID: 22063096
Authors: Street, T. O. - Lavery, L. A. - Verba, K. A. - Lee, C. T. - Mayer, M. P. - Agard, D. A.
Journal: J Mol Biol

The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.

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Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen.

Publication Date: 2012 Jan 6 PMID: 22063095
Authors: Feld, G. K. - Kintzer, A. F. - Tang, I. I. - Thoren, K. L. - Krantz, B. A.
Journal: J Mol Biol

The three protein components of anthrax toxin are nontoxic individually, but they form active holotoxin complexes upon assembly. The role of the protective antigen (PA) component of the toxin is to deliver two other enzyme components, lethal factor and edema factor, across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated; generally, cell surface activation is mediated by a furin family protease. Activated PA can then assemble into one of two noninterconverting oligomers, a homoheptamer and a homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA domain 4 (D4) to domain 2 with two different-length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 A resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of D4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin cleavage site and show that, for efficient processing, the furin site cannot be moved approximately 5 or 6 residues within the loop. We propose that there are different orientations of D4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

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Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope.

Publication Date: 2012 Jan 6 PMID: 22061265
Authors: Azoitei, M. L. - Ban, Y. E. - Julien, J. P. - Bryson, S. - Schroeter, A. - Kalyuzhniy, O. - Porter, J. R. - Adachi, Y. - Baker, D. - Pai, E. F. - Schief, W. R.
Journal: J Mol Biol

Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.

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High-Resolution Analysis of Zn(2+) Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography.

Publication Date: 2012 Jan 6 PMID: 22056344
Authors: Bobyr, E. - Lassila, J. K. - Wiersma-Koch, H. I. - Fenn, T. D. - Lee, J. J. - Nikolic-Hughes, I. - Hodgson, K. O. - Rees, D. C. - Hedman, B. - Herschlag, D.
Journal: J Mol Biol

Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn(2+) catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn(2+) coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29-A crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and NPP in the free-enzyme forms, with AMP and inorganic phosphate ground-state analogs and with vanadate transition-state analogs. These studies yielded average zinc-ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal-ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn(2+) catalytic site remains very similar between AP and NPP during the course of a reaction cycle.

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Structure of the Glycosyltransferase EryCIII in Complex with its Activating P450 Homologue EryCII.

Publication Date: 2012 Jan 6 PMID: 22056329
Authors: Moncrieffe, M. C. - Fernandez, M. J. - Spiteller, D. - Matsumura, H. - Gay, N. J. - Luisi, B. F. - Leadlay, P. F.
Journal: J Mol Biol

In the biosynthesis of the clinically important antibiotic erythromycin D, the glycosyltransferase (GT) EryCIII, in concert with its partner EryCII, attaches a nucleotide-activated sugar to the macrolide scaffold with high specificity. To understand the role of EryCII, we have determined the crystal structure of the EryCIII.EryCII complex at 3.1 A resolution. The structure reveals a heterotetramer with a distinctive, elongated quaternary organization. The EryCIII subunits form an extensive self-complementary dimer interface at the center of the complex, and the EryCII subunits lie on the periphery. EryCII binds in the vicinity of the putative macrolide binding site of EryCIII but does not make direct interactions with this site. Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT.

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DDX1 Is an RNA-Dependent ATPase Involved in HIV-1 Rev Function and Virus Replication.

Publication Date: 2012 Jan 6 PMID: 22051512
Authors: Edgcomb, S. P. - Carmel, A. B. - Naji, S. - Ambrus-Aikelin, G. - Reyes, J. R. - Saphire, A. C. - Gerace, L. - Williamson, J. R.
Journal: J Mol Biol

The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.

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