Proteins

Fold homology detection using sequence fragment composition profiles of proteins.

Publication Date: 2010 Oct PMID: 20635424
Authors: Solis, A. D. - Rackovsky, S. R.
Journal: Proteins

The effectiveness of sequence alignment in detecting structural homology among protein sequences decreases markedly when pairwise sequence identity is low (the so-called "twilight zone" problem of sequence alignment). Alternative sequence comparison strategies able to detect structural kinship among highly divergent sequences are necessary to address this need. Among them are alignment-free methods, which use global sequence properties (such as amino acid composition) to identify structural homology in a rapid and straightforward way. We explore the viability of using tetramer sequence fragment composition profiles in finding structural relationships that lie undetected by traditional alignment. We establish a strategy to recast any given protein sequence into a tetramer sequence fragment composition profile, using a series of amino acid clustering steps that have been optimized for mutual information. Our method has the effect of compressing the set of 160,000 unique tetramers (if using the 20-letter amino acid alphabet) into a more tractable number of reduced tetramers (approximately 15-30), so that a meaningful tetramer composition profile can be constructed. We test remote homology detection at the topology and fold superfamily levels using a comprehensive set of fold homologs, culled from the CATH database that share low pairwise sequence similarity. Using the receiver-operating characteristic measure, we demonstrate potentially significant improvement in using information-optimized reduced tetramer composition, over methods relying only on the raw amino acid composition or on traditional sequence alignment, in homology detection at or below the "twilight zone".

post to: CiteULike

TASSER_low-zsc: an approach to improve structure prediction using low z-score-ranked templates.

Publication Date: 2010 Oct PMID: 20635423
Authors: Pandit, S. B. - Skolnick, J.
Journal: Proteins

In a variety of threading methods, often poorly ranked (low z-score) templates have good alignments. Here, a new method, TASSER_low-zsc that identifies these low z-score-ranked templates to improve protein structure prediction accuracy, is described. The approach consists of clustering of threading templates by affinity propagation on the basis of structural similarity (thread_cluster) followed by TASSER modeling, with final models selected by using a TASSER_QA variant. To establish the generality of the approach, templates provided by two threading methods, SP(3) and SPARKS(2), are examined. The SP(3) and SPARKS(2) benchmark datasets consist of 351 and 357 medium/hard proteins (those with moderate to poor quality templates and/or alignments) of length < or =250 residues, respectively. For SP(3) medium and hard targets, using thread_cluster, the TM-scores of the best template improve by approximately 4 and 9% over the original set (without low z-score templates) respectively; after TASSER modeling/refinement and ranking, the best model improves by approximately 7 and 9% over the best model generated with the original template set. Moreover, TASSER_low-zsc generates 22% (43%) more foldable medium (hard) targets. Similar improvements are observed with low-ranked templates from SPARKS(2). The template clustering approach could be applied to other modeling methods that utilize multiple templates to improve structure prediction.

post to: CiteULike

i-Patch: interprotein contact prediction using local network information.

Publication Date: 2010 Oct PMID: 20635422
Authors: Hamer, R. - Luo, Q. - Armitage, J. P. - Reinert, G. - Deane, C. M.
Journal: Proteins

Biological processes are commonly controlled by precise protein-protein interactions. These connections rely on specific amino acids at the binding interfaces. Here we predict the binding residues of such interprotein complexes. We have developed a suite of methods, i-Patch, which predict the interprotein contact sites by considering the two proteins as a network, with residues as nodes and contacts as edges. i-Patch starts with two proteins, A and B, which are assumed to interact, but for which the structure of the complex is not available. However, we assume that for each protein, we have a reference structure and a multiple sequence alignment of homologues. i-Patch then uses the propensities of patches of residues to interact, to predict interprotein contact sites. i-Patch outperforms several other tested algorithms for prediction of interprotein contact sites. It gives 59% precision with 20% recall on a blind test set of 31 protein pairs. Combining the i-Patch scores with an existing correlated mutation algorithm, McBASC, using a logistic model gave little improvement. Results from a case study, on bacterial chemotaxis protein complexes, demonstrate that our predictions can identify contact residues, as well as suggesting unknown interfaces in multiprotein complexes.

post to: CiteULike

Structure of the amino-terminal domain from the cell-cycle regulator Swi6.

Publication Date: 2010 Oct PMID: 20635421
Authors: Taylor, I. A. - Goldstone, D. C. - Pala, P. - Haire, L. F. - Smerdon, S. J.
Journal: Proteins

post to: CiteULike

Fold versus sequence effects on the driving force for protein-mediated electron transfer.

Publication Date: 2010 Oct PMID: 20635418
Authors: Perrin, B. S. Jr - Ichiye, T.
Journal: Proteins

Electron transport chains composed of electron transfer reactions mainly between proteins provide fast efficient flow of energy in a variety of metabolic pathways. Reduction potentials are essential characteristics of the proteins because they determine the driving forces for the electron transfers. As both polar and charged groups from the backbone and side chains define the electrostatic environment, both the fold and the sequence will contribute. However, although the role of a specific sequence may be determined by experimental mutagenesis studies of reduction potentials, understanding the role of the fold by experiment is much more difficult. Here, continuum electrostatics and density functional theory calculations are used to analyze reduction potentials in [4Fe-4S] proteins. A key feature is that multiple homologous proteins in three different folds are compared: six high potential iron-sulfur proteins, four bacterial ferredoxins, and four nitrogenase iron proteins. Calculated absolute reduction potentials are shown to be in quantitative agreement with electrochemical reduction potentials. Calculations further demonstrate that the contribution of the backbone is larger than that of the side chains and is consistent for homologous proteins but differs for nonhomologous proteins, indicating that the fold is the major protein factor determining the reduction potential, whereas the specific amino acid sequence tunes the reduction potential for a given fold. Moreover, the fold contribution is determined mainly by the proximity of the redox site to the protein surface and the orientation of the dipoles of backbone near the redox site.

post to: CiteULike

Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase.

Publication Date: 2010 Oct PMID: 20635417
Authors: Chen, J. H. - Tsai, L. C. - Huang, H. C. - Shyur, L. F.
Journal: Proteins

We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TF-glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product beta-1,3-1,4-cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF-glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10-fold decrease of specific activity (11.2- to 67.4-fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17- to 4.3-fold increase of K(m) value when compared with TF-glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF-glucanase, by 2155-, 84.9-, 48.5-, 41.1-, and 19.1-fold, respectively; the five mutants showed the greatest changes in comparative energy DeltaDeltaG(b), with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X-ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF-glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites -3, -2, and -1 of CLTR/substrate. The overall globular structures in the wild-type and mutant F40I enzymes do not differ.

post to: CiteULike

Structure of the ATP-binding domain of Plasmodium falciparum Hsp90.

Publication Date: 2010 Oct PMID: 20635416
Authors: Corbett, K. D. - Berger, J. M.
Journal: Proteins

Hsp90 is an important cellular chaperone and attractive target for therapeutics against both cancer and infectious organisms. The Hsp90 protein from the parasite Plasmodium falciparum, the causative agent of malaria, is critical for this organism's survival; the anti-Hsp90 drug geldanamycin is toxic to P. falciparum growth. We have solved the structure of the N-terminal ATP-binding domain of P. falciparum Hsp90, which contains a principal drug-binding pocket, in both apo and ADP-bound states at 2.3 A resolution. The structure shows that P. falciparum Hsp90 is highly similar to human Hsp90, and likely binds agents such as geldanamycin in an identical manner. Our results should aid in the structural understanding of Hsp90-drug interactions in P. falciparum, and provide a scaffold for future drug-discovery efforts.

post to: CiteULike

The role of Buergi-Dunitz interactions in the structural stability of proteins.

Publication Date: 2010 Oct PMID: 20635415
Authors: Fufezan, C.
Journal: Proteins

Nonbonding interactions are essential for protein stability and maintenance of secondary structure. Their strength, however, is not always experimentally accessible. One example is the stability of collagen, which is in part due to Buergi-Duntiz or n --> pi* interactions between the peptide backbone atoms [DeRider et al., J Am Chem Soc 2002;124:2497-2505]. Here, the overall frequency of n --> pi* interactions in proteins has been investigated. The analysis of a nonredundant set of protein structures showed that 45.1% of all residues have a backbone conformation favoring a n --> pi* nucleophilic attack between the carbonyl oxygen of residue i - 1 and the carbonyl carbon of residue i. These residues form a substantial fraction of right- and left-handed alpha helices, 3(10) helices, pi helices, and hydrogen bonded turns. Simulations showed that there are only four regions in Ramachandran space that favor backbone n(i-1) --> pi(i) (*) interactions and these Phi, Psi combinations are observed with high frequencies in the nonredundant protein structure set. Analysis of carbonyl carbon displacements out of the peptide plane in ultra-high resolution protein structures indeed reveals the presence of the Buergi-Dunitz trajectory. The Buergi-Dunitz interaction thus appears to play an important and general role in protein structure stability that has not hitherto been fully explored.

post to: CiteULike

Application of biasing-potential replica-exchange simulations for loop modeling and refinement of proteins in explicit solvent.

Publication Date: 2010 Oct PMID: 20635348
Authors: Kannan, S. - Zacharias, M.
Journal: Proteins

Comparative or homology modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. Depending on the target-template similarity these model structures may contain regions of limited structural accuracy. In principle, molecular dynamics (MD) simulations can be used to refine protein model structures and also to model loop regions that connect structurally conserved regions but it is limited by the currently accessible simulation time scales. A recently developed biasing potential replica exchange (BP-REMD) method was used to refine loops and complete decoy protein structures at atomic resolution including explicit solvent. In standard REMD simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. In a BP-REMD simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. The method requires much fewer replicas for efficient sampling compared with T-REMD. Application of the approach to several protein loops indicated improved conformational sampling of backbone dihedral angle of loop residues compared to conventional MD simulations. BP-REMD refinement simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional MD simulations.

post to: CiteULike

Crystal structure of a truncated urease accessory protein UreF from Helicobacter pylori.

Publication Date: 2010 Oct PMID: 20635345
Authors: Lam, R. - Romanov, V. - Johns, K. - Battaile, K. P. - Wu-Brown, J. - Guthrie, J. L. - Hausinger, R. P. - Pai, E. F. - Chirgadze, N. Y.
Journal: Proteins

Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG, which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C-terminal truncated UreF from H. pylori (residues 1-233), the first UreF structure to be determined, at 1.55 A resolution using SAD methods. UreF forms a dimer in vitro and adopts an all-helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance.

post to: CiteULike

Dry molten globule intermediates and the mechanism of protein unfolding.

Publication Date: 2010 Oct PMID: 20635344
Authors: Baldwin, R. L. - Frieden, C. - Rose, G. D.
Journal: Proteins

New experimental results show that either gain or loss of close packing can be observed as a discrete step in protein folding or unfolding reactions. This finding poses a significant challenge to the conventional two-state model of protein folding. Results of interest involve dry molten globule (DMG) intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the DMG state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form DMGs as the first step of unfolding, suggesting that such an intermediate may well be commonplace in both folding and unfolding. Data from the literature show that peptide amide protons are protected in the DMG, indicating that backbone structure is intact despite loss of side-chain close packing. Other complementary evidence shows that secondary structure formation provides a major source of compaction during folding. In our model, the major free-energy barrier separating unfolded from native states usually occurs during the transition between the unfolded state and the DMG. The absence of close packing at this barrier provides an explanation for why phi-values, derived from a Bronsted-Leffler plot, depend primarily on structure at the mutational site and not on specific side-chain interactions. The conventional two-state folding model breaks down when there are DMG intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding.

post to: CiteULike

Binding of nonsteroidal anti-inflammatory drugs to Abeta fibril.

Publication Date: 2010 Oct PMID: 20635343
Authors: Takeda, T. - Chang, W. E. - Raman, E. P. - Klimov, D. K.
Journal: Proteins

Nonsteroidal anti-inflammatory drugs are considered as potential therapeutic agents against Alzheimer's disease. Using replica exchange molecular dynamics and atomistic implicit solvent model, we studied the mechanisms of binding of naproxen and ibuprofen to the Abeta fibril derived from solid-state NMR measurements. The binding temperature of naproxen is found to be almost 40 K higher than of ibuprofen implicating higher binding affinity of naproxen. The key factor, which enhances naproxen binding, is strong interactions between ligands bound to the surface of the fibril. The naphthalene ring in naproxen appears to provide a dominant contribution to ligand-ligand interactions. In contrast, ligand-fibril interactions cannot explain differences in the binding affinities of naproxen and ibuprofen. The concave fibril edge with the groove is identified as the primary binding location for both ligands. We show that confinement of the ligands to the groove facilitates ligand-ligand interactions that lowers the energy of the ligands bound to the concave edge compared with those bound to the convex edge. Our simulations appear to provide microscopic rationale for the differing binding affinities of naproxen and ibuprofen observed experimentally.

post to: CiteULike

Gain of local structure in an amphipathic peptide does not require a specific tertiary framework.

Publication Date: 2010 Oct PMID: 20607854
Authors: Roman, E. A. - Rosi, P. - Gonzalez Lebrero, M. C. - Wuilloud, R. - Gonzalez Flecha, F. L. - Delfino, J. M. - Santos, J.
Journal: Proteins

In this work, we studied how an amphipathic peptide of the surface of the globular protein thioredoxin, TRX94-108, acquires a native-like structure when it becomes involved in an apolar interaction network. We designed peptide variants where the tendency to form alpha-helical conformation is modulated by replacing each of the leucine amino acid residues by an alanine. The induction of structure caused by sodium dodecyl sulfate (SDS) binding was studied by capillary zone electrophoresis, circular dichroism, DOSY-NMR, and molecular dynamics simulations (MDS). In addition, we analyzed the strength of the interaction between a C18 RP-HPLC matrix and the peptides. The results presented here reveal that (a) critical elements in the sequence of the wild-type peptide stabilize a SDS/peptide supramolecular cluster; (b) the hydrophobic nature of the interaction between SDS molecules and the peptide constrains the ensemble of conformations; (c) nonspecific apolar surfaces are sufficient to stabilize peptide secondary structure. Remarkably, MDS shed light on a contact network formed by a limited number of SDS molecules that serves as a structural scaffold preserving the helical conformation of this module. This mechanism might prevail when a peptide with low helical propensity is involved in structure consolidation. We suggest that folding of peptides sharing this feature does not require a preformed tightly-packed protein core. Thus, the formation of specific tertiary interactions would be the consequence of peptide folding and not its cause. In this scenario, folding might be thought of as a process that includes unspecific rounds of structure stabilization guiding the protein to the native state.

post to: CiteULike

The measured and calculated affinity of methyl- and methoxy-substituted benzoquinones for the Q(A) site of bacterial reaction centers.

Publication Date: 2010 Sep PMID: 20607696
Authors: Zheng, Z. - Dutton, P. L. - Gunner, M. R.
Journal: Proteins

Quinones play important roles in mitochondrial and photosynthetic energy conversion acting as intramembrane, mobile electron, and proton carriers between catalytic sites in various electron transfer proteins. They display different affinity, selectivity, functionality, and exchange dynamics in different binding sites. The computational analysis of quinone binding sheds light on the requirements for quinone affinity and specificity. The affinities of 10 oxidized, neutral benzoquinones were measured for the high affinity Q(A) site in the detergent-solubilized Rhodobacter sphaeroides bacterial photosynthetic reaction center. Multiconformation Continuum Electrostatics was then used to calculate their relative binding free energies by grand canonical Monte Carlo sampling with a rigid protein backbone, flexible ligand, and side chain positions and protonation states. Van der Waals and torsion energies, Poisson-Boltzmann continuum electrostatics, and accessible surface area-dependent ligand-solvent interactions are considered. An initial, single cycle of GROMACS backbone optimization improves the match with experiment as do coupled-ligand and side-chain motions. The calculations match experiment with an root mean square deviation (RMSD) of 2.29 and a slope of 1.28. The affinities are dominated by favorable protein-ligand van der Waals rather than electrostatic interactions. Each quinone appears in a closely clustered set of positions. Methyl and methoxy groups move into the same positions as found for the native quinone. Difficulties putting methyls into methoxy sites are observed. Calculations using a solvent-accessible surface area-dependent implicit van der Waals interaction smoothed out small clashes, providing a better match to experiment with a RMSD of 0.77 and a slope of 0.97.

post to: CiteULike

Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model.

Publication Date: 2010 Aug 15 PMID: 20602461
Authors: Tekpinar, M. - Zheng, W.
Journal: Proteins

The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions of various biological nanomachines. Coarse-grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse-grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP-driven transitions in three important biological nanomachines (myosin, F(1) ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines.

post to: CiteULike

The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

Publication Date: 2010 Aug 15 PMID: 20602460
Authors: Doucet, N. - Jayasundera, T. B. - Simonovic, M. - Loria, J. P.
Journal: Proteins

Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme.

post to: CiteULike

Structure and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA.

Publication Date: 2010 Aug 15 PMID: 20602459
Authors: Agarwal, S. - Hong, D. - Desai, N. K. - Sazinsky, M. H. - Arguello, J. M. - Rosenzweig, A. C.
Journal: Proteins

The Cu(+)-ATPase CopA from Archaeoglobus fulgidus belongs to the P(1B) family of the P-type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P(1B-1)-type ATPases is the presence of soluble metal binding domains at the N-terminus (N-MBDs). The N-MBDs exhibit a conserved ferredoxin-like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N-MBDs enable Cu(+) regulation of turnover rates apparently through Cu-sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N-terminal MBD and a C-terminal MBD (C-MBD). The functional role of the unique C-MBD has not been established. Here, we report the crystal structure of the apo, oxidized C-MBD to 2.0 A resolution. In the structure, two C-MBD monomers form a domain-swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C-MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A-domain), has been investigated. Interestingly, the C-MBD interacts specifically with both of these domains, independent of the presence of Cu(+) or nucleotides. These data reinforce the uniqueness of the C-MBD and suggest a distinct structural role for the C-MBD in CopA transport.

post to: CiteULike

Analysis of Ca2+/Mg2+ selectivity in alpha-lactalbumin and Ca(2+)-binding lysozyme reveals a distinct Mg(2+)-specific site in lysozyme.

Publication Date: 2010 Sep PMID: 20602456
Authors: Permyakov, S. E. - Khokhlova, T. I. - Uversky, V. N. - Permyakov, E. A.
Journal: Proteins

The triggering of Ca(2+) signaling pathways relies on Ca(2+)/Mg(2+) specificity of proteins mediating these pathways. Two homologous milk Ca(2+)-binding proteins, bovine alpha-lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest "four-state" scheme of metal- and temperature-induced structural changes in a protein. The association of Ca(2+)/Mg(2+) by native proteins is entropy-driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca(2+) and Mg(2+), due to low thermal stabilities of their apo-forms and relatively high unfavorable enthalpies of Mg(2+) association. The ratios of their apparent affinities to Ca(2+) and Mg(2+), being unusually high at low temperatures (5.3-6.5 orders of magnitude), reach the values inherent to classical EF-hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca(2+) and Mg(2+) binding with experimental data strongly suggests that the association of Ca(2+) and Mg(2+) ions with bLA is a competitive process, whereas the primary Mg(2+) site of EQL is different from its Ca(2+)-binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near-UV region accompanying Mg(2+) and Ca(2+) association. The Ca(2+)/Mg(2+) selectivity of Mg(2+)-site of EQL is below an order of magnitude. EQL exhibits a distinct Mg(2+)-specific site, probably arising as an adaptation to the extracellular environment.

post to: CiteULike

Two-state conformational equilibrium in the Par-4 leucine zipper domain.

Publication Date: 2010 Aug 15 PMID: 20602362
Authors: Schwalbe, M. - Dutta, K. - Libich, D. S. - Venugopal, H. - Claridge, J. K. - Gell, D. A. - Mackay, J. P. - Edwards, P. J. - Pascal, S. M.
Journal: Proteins

Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par-4 at neutral pH. Further, we have investigated the properties of the Par-4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment-dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium.

post to: CiteULike

Diterpene cyclases and the nature of the isoprene fold.

Publication Date: 2010 Aug 15 PMID: 20602361
Authors: Cao, R. - Zhang, Y. - Mann, F. M. - Huang, C. - Mukkamala, D. - Hudock, M. P. - Mead, M. E. - Prisic, S. - Wang, K. - Lin, F. Y. - Chang, T. K. - Peters, R. J. - Oldfield, E.
Journal: Proteins

The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain approximately 20 alpha-helices and the same conserved "QW" and DxDD motifs as in triterpene cyclases, indicating the presence of a betagamma barrel structure. Plant diterpene cyclases have a similar catalytic motif and betagamma-domain structure together with a third, alpha-domain, forming an alphabetagamma structure, and in H(+)-initiated cyclases, there is an EDxxD-like Mg(2+)/diphosphate binding motif located in the gamma-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (betagamma) bacterial triterpene cyclases to (betagamma) bacterial and thence to (alphabetagamma) plant diterpene cyclases.

post to: CiteULike

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy.

Publication Date: 2010 Aug 15 PMID: 20602360
Authors: Lawrenz, M. - Wereszczynski, J. - Amaro, R. - Walker, R. - Roitberg, A. - McCammon, J. A.
Journal: Proteins

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium-bound and calcium-free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can "clamp" the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods.

post to: CiteULike

A molecular dynamics study of the early stages of amyloid-beta(1-42) oligomerization: the role of lipid membranes.

Publication Date: 2010 Aug 15 PMID: 20602359
Authors: Davis, C. H. - Berkowitz, M. L.
Journal: Proteins

As research progresses toward understanding the role of the amyloid-beta (Abeta) peptide in Alzheimer's disease, certain aspects of the aggregation process for Abeta are still not clear. In particular, the accepted constitution of toxic aggregates in neurons has shifted toward small oligomers. However, the process of forming these oligomers in cells is also not full clear. Even more interestingly, it has been implied that cell membranes, and, in particular, anionic lipids within those membranes, play a key role in the progression of Abeta aggregation, but the exact nature of the Abeta-membrane interaction in this process is unknown. In this work, we use a thermodynamic cycle and umbrella sampling molecular dynamics to investigate dimerization of the 42-residue Abeta peptide on model zwitterionic dipalmitoylphosphatidylcholine (DPPC) or model anionic dioleoylphosphatidylserine (DOPS) bilayer surfaces. We determined that Abeta dimerization was strongly favored through interactions with the DOPS bilayer. Further, our calculations showed that the DOPS bilayer promoted strong protein-protein interactions within the Abeta dimer, whereas DPPC favored strong protein-lipid interactions. By promoting dimer formation and subsequent dimer release into the solvent, the DOPS bilayer acts as a catalyst in Abeta aggregation.

post to: CiteULike

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation.

Publication Date: 2010 Aug 15 PMID: 20602358
Authors: Auer, S. - Kashchiev, D.
Journal: Proteins

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation.

post to: CiteULike

Solution NMR structure of Lin0431 protein from Listeria innocua reveals high structural similarity with domain II of bacterial transcription antitermination protein NusG.

Publication Date: 2010 Aug 15 PMID: 20602357
Authors: Tang, Y. - Xiao, R. - Ciccosanti, C. - Janjua, H. - Lee, D. Y. - Everett, J. K. - Swapna, G. V. - Acton, T. B. - Rost, B. - Montelione, G. T.
Journal: Proteins

post to: CiteULike

The catch bond mechanism between von Willebrand factor and platelet surface receptors investigated by molecular dynamics simulations.

Publication Date: 2010 Aug 15 PMID: 20602356
Authors: Interlandi, G. - Thomas, W.
Journal: Proteins

The multi-domain protein von Willebrand factor is crucial in the blood coagulation process at high shear. The A1 domain binds to the platelet surface receptor glycoprotein Ibalpha (GpIb alpha) and this interaction is known to be strengthened by tensile force. The molecular mechanism behind this observation was investigated here by molecular dynamics simulations. The results suggest that the proteins unbind through two distinct pathways depending whether a high-tensile force is applied or whether unbinding happens through thermal fluctuations. In the high-force unbinding pathway the A1 domain was observed to rotate away from the C-terminus of GpIb alpha. In contrast, during thermal unbinding the A1 domain rotated in the opposite direction as in the high-force pathway and the distance between the terminii of A1 and the GpIb alpha C-terminus shortened. This shortening was reduced and the lifetime of the bond extended if a moderate tensile force was applied across the complex. This suggests that the thermal unbinding pathway is inhibited by a moderate tensile force which is in agreement with the catch bond property shown previously in single molecule experiments. A designed mutant of GpIb alpha is suggested here in order to test in vitro the thermal unbinding pathway observed in silico.

post to: CiteULike

Pentacoordinated phosphorus revisited by high-level QM/MM calculations.

Publication Date: 2010 Aug 15 PMID: 20602355
Authors: Marcos, E. - Field, M. J. - Crehuet, R.
Journal: Proteins

Enzymes catalyzing phosphoryl transfer reactions are extremely efficient and are involved in crucial biochemical processes. The mechanisms of these enzymes are complex due to the diversity of substrates that are involved. The reaction can proceed through a pentacoordinated phosphorus species that is either a stable intermediate or a transition state (TS). Because of this, the first X-ray structure of a pentacoordinated phosphorus intermediate in the beta-phosphoglucomutase enzyme aroused great interest but also much controversy. To provide new insights into the nature of that structure, we have determined the reaction path of the phosphorylation step using high-level QM/MM calculations, and have also calculated the geometry of a complex with a transition state analogue (TSA) that has been suggested to be the actual species in the crystal. The protein crystalline environment has been modeled so as to mimic the experimental conditions. We conclude that the pentacoordinated phosphorus formed in this enzyme is not a stable species but a TS, which gives an activation energy for phosphorylation in agreement with kinetic results. We also show that the TSA is a good mimic of the true TS. We have performed a new crystallographic refinement of the original diffraction map of the pentacoordinated phosphorus structure with the MgF(3)(-) TSA. The new fit improves significantly with respect to the original one, which strongly supports that Allen and coworkers wrongly assigned the X-ray structure to a pentavalent phosphorane.

post to: CiteULike

Antibodies as a model system for comparative model refinement.

Publication Date: 2010 Aug 15 PMID: 20602354
Authors: Sellers, B. D. - Nilmeier, J. P. - Jacobson, M. P.
Journal: Proteins

Predicting the conformations of loops is a critical aspect of protein comparative (homology) modeling. Despite considerable advances in developing loop prediction algorithms, refining loops in homology models remains challenging. In this work, we use antibodies as a model system to investigate strategies for more robustly predicting loop conformations when the protein model contains errors in the conformations of side chains and protein backbone surrounding the loop in question. Specifically, our test system consists of partial models of antibodies in which the "scaffold" (i.e., the portion other than the complementarity determining region, CDR, loops) retains native backbone conformation, whereas the CDR loops are predicted using a combination of knowledge-based modeling (H1, H2, L1, L2, and L3) and ab initio loop prediction (H3). H3 is the most variable of the CDRs. Using a previously published method, a test set of 10 shorter H3 loops (5-7 residues) are predicted to an average backbone (N-C alpha-C-O) RMSD of 2.7 A while 11 longer loops (8-9 residues) are predicted to 5.1 A, thus recapitulating the difficulties in refining loops in models. By contrast, in control calculations predicting the same loops in crystal structures, the same method reconstructs the loops to an average of 0.5 and 1.4 A for the shorter and longer loops, respectively. We modify the loop prediction method to improve the ability to sample near-native loop conformations in the models, primarily by reducing the sensitivity of the sampling to the loop surroundings, and allowing the other CDR loops to optimize with the H3 loop. The new method improves the average accuracy significantly to 1.3 A RMSD and 3.1 A RMSD for the shorter and longer loops, respectively. Finally, we present results predicting 8-10 residue loops within complete comparative models of five nonantibody proteins. While anecdotal, these mixed, full-model results suggest our approach is a promising step toward more accurately predicting loops in homology models. Furthermore, while significant challenges remain, our method is a potentially useful tool for predicting antibody structures based on a known Fv scaffold.

post to: CiteULike

Validation of archived chemical shifts through atomic coordinates.

Publication Date: 2010 Aug 15 PMID: 20602353
Authors: Rieping, W. - Vranken, W. F.
Journal: Proteins

The public archives containing protein information in the form of NMR chemical shift data at the BioMagResBank (BMRB) and of 3D structure coordinates at the Protein Data Bank are continuously expanding. The quality of the data contained in these archives, however, varies. The main issue for chemical shift values is that they are determined relative to a reference frequency. When this reference frequency is set incorrectly, all related chemical shift values are systematically offset. Such wrongly referenced chemical shift values, as well as other problems such as chemical shift values that are assigned to the wrong atom, are not easily distinguished from correct values and effectively reduce the usefulness of the archive. We describe a new method to correct and validate protein chemical shift values in relation to their 3D structure coordinates. This method classifies atoms using two parameters: the per-atom solvent accessible surface area (as calculated from the coordinates) and the secondary structure of the parent amino acid. Through the use of Gaussian statistics based on a large database of 3220 BMRB entries, we obtain per-entry chemical shift corrections as well as Z scores for the individual chemical shift values. In addition, information on the error of the correction value itself is available, and the method can retain only dependable correction values. We provide an online resource with chemical shift, atom exposure, and secondary structure information for all relevant BMRB entries (http://www.ebi.ac.uk/pdbe/nmr/vasco) and hope this data will aid the development of new chemical shift-based methods in NMR.

post to: CiteULike

Structural studies of mutant forms of the PQQ-forming enzyme PqqC in the presence of product and substrate.

Publication Date: 2010 Aug 15 PMID: 20602352
Authors: Puehringer, S. - RoseFigura, J. - Metlitzky, M. - Toyama, H. - Klinman, J. P. - Schwarzenbacher, R.
Journal: Proteins

Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial cofactor in numerous alcohol dehydrogenases including methanol dehydrogenase and glucose dehydrogenase. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF and proceeds by an unknown pathway. PqqC is one of two metal free oxidases of known structure and catalyzes the last step of PQQ biogenesis which involves a ring closure and an eight-electron oxidation of the substrate [3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9- dicarboxylic acid (AHQQ)]. PqqC has 14 conserved active site residues, which have previously been shown to be in close contact with bound PQQ. Herein, we describe the structures of three PqqC active site variants, H154S, Y175F, and the double mutant R179S/Y175S. The H154S crystal structure shows that, even with PQQ bound, the enzyme is still in the "open" conformation with helices alpha5b and alpha6 unfolded and the active site solvent accessible. The Y175F PQQ complex crystal structure reveals the closed conformation indicating that Y175 is not required for the conformational change. The R179S/Y175S AHQQ complex crystal structure is the most mechanistically informative, indicating an open conformation with a reaction intermediate trapped in the active site. The intermediate seen in R179S/Y175S is tricyclic but nonplanar, implying that it has not undergone oxidation. These studies implicate a stepwise process in which substrate binding leads to the generation of the closed protein conformation, with the latter playing a critical role in O(2) binding and catalysis.

post to: CiteULike

Crystal structure of archaemetzincin amza from Methanopyrus kandleri at 1.5 A resolution.

Publication Date: 2010 Sep PMID: 20597090
Authors: Waltersperger, S. - Widmer, C. - Wang, M. - Baumann, U.
Journal: Proteins

post to: CiteULike

Role of partial protein unfolding in alcohol-induced protein aggregation.

Publication Date: 2010 Sep PMID: 20597088
Authors: Singh, S. M. - Cabello-Villegas, J. - Hutchings, R. L. - Mallela, K. M.
Journal: Proteins

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.

post to: CiteULike

CRK: an evolutionary approach for distinguishing biologically relevant interfaces from crystal contacts.

Publication Date: 2010 Sep PMID: 20589644
Authors: Scharer, M. A. - Grutter, M. G. - Capitani, G.
Journal: Proteins

Protein crystals contain two different types of interfaces: biologically relevant ones, observed in protein-protein complexes and oligomeric proteins, and nonspecific ones, corresponding to crystal lattice contacts. Because of the increasing complexity of the objects being tackled in structural biology, distinguishing biological contacts from crystal contacts is not always a trivial task and can lead to wrong interpretation of macromolecular structures. We devised an approach (CRK, core-rim K(a)/K(s) ratio) for distinguishing biologically relevant interfaces from nonspecific ones. Given a protein-protein interface, CRK finds a set of homologs to the sequences of the proteins involved in the interface, retrieves and aligns the corresponding coding sequences, on which it carries out a residue-by-residue K(a)/K(s) ratio (omega) calculation. It divides interface residues into a "rim" and a "core" set and analyzes the selection pressure on the residues belonging to the two sets. We developed and tested CRK on different datasets and test cases, consisting of biologically relevant contacts, nonspecific ones or of both types. The method proves very effective in distinguishing the two categories of interfaces, with an overall accuracy rate of 84%. As it relies on different principles when compared with existing tools, CRK is optimally suited to be used in combination with them. In addition, CRK has potential applications in the validation of structures of oligomeric proteins and protein complexes.

post to: CiteULike

Crystal structures of the apo and GDP-bound forms of a cupin-like protein BbDUF985 from Branchiostoma belcheri tsingtauense.

Publication Date: 2010 Sep PMID: 20589641
Authors: Du, Y. - He, Y. X. - Gaowa, S. - Zhang, X. - Chen, Y. - Zhang, S. C. - Zhou, C. Z.
Journal: Proteins

post to: CiteULike

New hypotheses about the structure-function of proprotein convertase subtilisin/kexin type 9: analysis of the epidermal growth factor-like repeat A docking site using WaterMap.

Publication Date: 2010 Sep PMID: 20589640
Authors: Pearlstein, R. A. - Hu, Q. Y. - Zhou, J. - Yowe, D. - Levell, J. - Dale, B. - Kaushik, V. K. - Daniels, D. - Hanrahan, S. - Sherman, W. - Abel, R.
Journal: Proteins

LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein-ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-of-function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins ("hydration sites"). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast k(on) and entropically driven thermodynamics observed for PCSK9-EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast k(off) observed for EGF-A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k(off) observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex.

post to: CiteULike

Similarity of cytochrome c oxidases in different organisms.

Publication Date: 2010 Sep PMID: 20589635
Authors: Popovic, D. M. - Leontyev, I. V. - Beech, D. G. - Stuchebrukhov, A. A.
Journal: Proteins

Most of biological oxygen reduction is catalyzed by the heme-copper oxygen reductases. These enzymes are redox-driven proton pumps that take part in generating the proton gradient in both prokaryotes and mitochondria that drives synthesis of ATP. The enzymes have been divided into three evolutionarily-related groups: the A-, B-, and C-families. Recent comparative studies suggest that all oxygen reductases perform the same chemistry for oxygen reduction and comprise the same essential elements of the proton pumping mechanism, such as the proton loading and kinetic gating sites, which, however, appear to be different in different families. All species of the A-family, however, demonstrate remarkable similarity of the central processing unit of the enzyme, as revealed by their recent crystal structures. Here we demonstrate that cytochrome c oxidases (CcO) of such diverse organisms as a mammal (bovine heart mitochondrial CcO), photosynthetic bacteria (Rhodobacter sphaeroides CcO), and soil bacteria (Paracoccus denitrificans CcO) are not only structurally similar, but almost identical in microscopic electrostatics and thermodynamics properties of their key amino-acids. By using pK(a) calculations of some of the key residues of the catalytic site, D- and K- proton input, and putative proton output channels of these three different enzymes, we demonstrate that the microscopic properties of key residues are almost identical, which strongly suggests the same mechanism in these species. The quantitative precision with which the microscopic physical properties of these enzymes have remained constant despite different evolutionary routes undertaken is striking.

post to: CiteULike

Exploring the role of the phospholipid ligand in endothelial protein C receptor: a molecular dynamics study.

Publication Date: 2010 Sep PMID: 20589634
Authors: Chiappori, F. - Merelli, I. - Milanesi, L. - Rovida, E.
Journal: Proteins

Endothelial protein C receptor (EPCR) is a CD1-like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti-inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen-presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC gamma-carboxyglutamic acid (Gla) domain binding. However, the lipid-free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand-free conformations. Compared with the bound form, unbound structures showed a narrowing of the A' pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution-induced conformational changes affecting helices around the A' pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests.

post to: CiteULike

Consistent refinement of submitted models at CASP using a knowledge-based potential.

Publication Date: 2010 Sep PMID: 20589633
Authors: Chopra, G. - Kalisman, N. - Levitt, M.
Journal: Proteins

Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, critical assessment of techniques for protein structure prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge-based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177-3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol.

post to: CiteULike

Comparing the folding free-energy landscapes of Abeta42 variants with different aggregation properties.

Publication Date: 2010 Sep PMID: 20589631
Authors: Mitternacht, S. - Staneva, I. - Hard, T. - Irback, A.
Journal: Proteins

The properties of the amyloid-beta peptide that lead to aggregation associated with Alzheimer's disease are not fully understood. This study aims at identifying conformational differences among four variants of full-length Abeta42 that are known to display very different aggregation properties. By extensive all-atom Monte Carlo simulations, we find that a variety of beta-sheet structures with distinct turns are readily accessible for full-length Abeta42. In the simulations, wild type (WT) Abeta42 preferentially populates two major classes of conformations, either extended with high beta-sheet content or more compact with lower beta-sheet content. The three mutations studied alter the balance between these classes. Strong mutational effects are observed in a region centered at residues 23-26, where WT Abeta42 tends to form a turn. The aggregation-accelerating E22G mutation associated with early onset of Alzheimer's disease makes this turn region conformationally more diverse, whereas the aggregation-decelerating F20E mutation has the reverse effect, and the E22G/I31E mutation reduces the turn population. Comparing results for the four Abeta42 variants, we identify specific conformational properties of residues 23-26 that might play a key role in aggregation.

post to: CiteULike

On the pH-optimum of activity and stability of proteins.

Publication Date: 2010 Sep PMID: 20589630
Authors: Talley, K. - Alexov, E.
Journal: Proteins

Biological macromolecules evolved to perform their function in specific cellular environment (subcellular compartments or tissues); therefore, they should be adapted to the biophysical characteristics of the corresponding environment, one of them being the characteristic pH. Many macromolecular properties are pH dependent, such as activity and stability. However, only activity is biologically important, while stability may not be crucial for the corresponding reaction. Here, we show that the pH-optimum of activity (the pH of maximal activity) is correlated with the pH-optimum of stability (the pH of maximal stability) on a set of 310 proteins with available experimental data. We speculate that such a correlation is needed to allow the corresponding macromolecules to tolerate small pH fluctuations that are inevitable with cellular function. Our findings rationalize the efforts of correlating the pH of maximal stability and the characteristic pH of subcellular compartments, as only pH of activity is subject of evolutionary pressure. In addition, our analysis confirmed the previous observation that pH-optimum of activity and stability are not correlated with the isoelectric point, pI, or with the optimal temperature.

post to: CiteULike

Species specificity of the complement inhibitor compstatin investigated by all-atom molecular dynamics simulations.

Publication Date: 2010 Sep PMID: 20589629
Authors: Tamamis, P. - Morikis, D. - Floudas, C. A. - Archontis, G.
Journal: Proteins

The development of compounds to regulate the activation of the complement system in non-primate species is of profound interest because it can provide models for human diseases. The peptide compstatin inhibits protein C3 in primate mammals and is a potential therapeutic agent against unregulated activation of complement in humans but is inactive against nonprimate species. Here, we elucidate this species specificity of compstatin by molecular dynamics simulations of complexes between the most potent natural compstatin analog and human or rat C3. The results are compared against an experimental conformation of the human complex, determined recently by X-ray diffraction at 2.4-A resolution. The human complex simulations provide information on the relative contributions to stability of specific C3 and compstatin residues. In the rat simulations, the protein undergoes reproducible conformational changes, which eliminate or weaken specific interactions and reduce the complex stability. The simulation insights can be used to design improved compstatin-based inhibitors for human C3 and active inhibitors against lower mammals.

post to: CiteULike

Structural comparison and classification of alpha-helical transmembrane domains based on helix interaction patterns.

Publication Date: 2010 Sep PMID: 20552684
Authors: Fuchs, A. - Frishman, D.
Journal: Proteins

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three-dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies alpha-helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of alpha-helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences.

post to: CiteULike

Evidence for specific subunit distribution and interactions in the quaternary structure of alpha-crystallin.

Publication Date: 2010 Aug 15 PMID: 20535821
Authors: Morris, A. M. - Aquilina, J. A.
Journal: Proteins

The quaternary structure of alpha-crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision-induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of alpha-crystallin. For total alpha-crystallin isolated directly from fetal calf lens using size-based chromatography, the alphaB-crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the alphaA-crystallin subunits. Furthermore, upon mixing molar equivalents of purified alphaA- and alphaB-crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the alphaA- and alphaB-crystallin homo-oligomers was comparable, indicating that strength of the alphaA:alphaA, and alphaB:alphaB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of alphaB-crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C-terminus of alphaA-crystallin was more susceptible to proteolytic attack in homo-oligomers than that of alphaB-crystallin. As subunit exchange proceeded, proteolysis of the alphaA-crystallin C-terminus increased, indicating that in the hetero-oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the alpha-crystallin domains and C-terminal extensions of subunits within the alpha-crystallin assembly. In particular, we propose that the palindromic IPI motif of alphaB-crystallin gives rise to two orientations of the C-terminus.

post to: CiteULike