Proteins

A universal pathway for amyloid nucleus and precursor formation for insulin.

Publication Date: 2008 Jul 24 PMID: 18655073
Authors: Nayak, A. - Sorci, M. - Krueger, S. - Belfort, G.
Journal: Proteins

To help identify the etiological agents for amyloid-related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H(2)O- and D(2)O-based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end-on-end association of dimers to form three-dimer nuclei, and then exhibits broadening or side-on-side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Structure based mechanism of the Ca(2+)-induced release of coelenterazine from the Renilla binding protein.

Publication Date: 2008 Jul 24 PMID: 18655070
Authors: Stepanyuk, G. A. - Liu, Z. J. - Vysotski, E. S. - Lee, J. - Rose, J. P. - Wang, B. C.
Journal: Proteins

The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation.

Publication Date: 2008 Jul 24 PMID: 18655068
Authors: Straatsma, T. P. - Soares, T. A.
Journal: Proteins

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Optimal data collection for correlated mutation analysis.

Publication Date: 2008 Jul 24 PMID: 18655065
Authors: Ashkenazy, H. - Unger, R. - Kliger, Y.
Journal: Proteins

The main objective of correlated mutation analysis (CMA) is to predict intraprotein residue-residue interactions from sequence alone. Despite considerable progress in algorithms and computer capabilities, the performance of CMA methods remains quite low. Here we examine whether, and to what extent, the quality of CMA methods depends on the sequences that are included in the multiple sequence alignment (MSA). The results revealed a strong correlation between the number of homologs in an MSA and CMA prediction strength. Furthermore, many of the current methods include only orthologs in the MSA, we found that it is beneficial to include both orthologs and paralogs in the MSA. Remarkably, even remote homologs contribute to the improved accuracy. Based on our findings we put forward an automated data collection procedure, with a minimal coverage of 50% between the query protein and its orthologs and paralogs. This procedure improves accuracy even in the absence of manual curation. In this era of massive sequencing and exploding sequence data, our results suggest that correlated mutation-based methods have not reached their inherent performance limitations and that the role of CMA in structural biology is far from being fulfilled. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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PFP: Automated prediction of gene ontology functional annotations with confidence scores using protein sequence data.

Publication Date: 2008 Jul 24 PMID: 18655063
Authors: Hawkins, T. - Chitale, M. - Luban, S. - Kihara, D.
Journal: Proteins

Protein function prediction is a central problem in bioinformatics, increasing in importance recently due to the rapid accumulation of biological data awaiting interpretation. Sequence data represents the bulk of this new stock and is the obvious target for consideration as input, as newly sequenced organisms often lack any other type of biological characterization. We have previously introduced PFP (Protein Function Prediction) as our sequence-based predictor of Gene Ontology (GO) functional terms. PFP interprets the results of a PSI-BLAST search by extracting and scoring individual functional attributes, searching a wide range of E-value sequence matches, and utilizing conventional data mining techniques to fill in missing information. We have shown it to be effective in predicting both specific and low-resolution functional attributes when sufficient data is unavailable. Here we describe (1) significant improvements to the PFP infrastructure, including the addition of prediction significance and confidence scores, (2) a thorough benchmark of performance and comparisons to other related prediction methods, and (3) applications of PFP predictions to genome-scale data. We applied PFP predictions to uncharacterized protein sequences from 15 organisms. Among these sequences, 60-90% could be annotated with a GO molecular function term at high confidence (>/=80%). We also applied our predictions to the protein-protein interaction network of the Malaria plasmodium (Plasmodium falciparum). High confidence GO biological process predictions (>/=90%) from PFP increased the number of fully enriched interactions in this dataset from 23% of interactions to 94%. Our benchmark comparison shows significant performance improvement of PFP relative to GOtcha, InterProScan, and PSI-BLAST predictions. This is consistent with the performance of PFP as the overall best predictor in both the AFP-SIG '05 and CASP7 function (FN) assessments. PFP is available as a web service at http://dragon.bio.purdue.edu/pfp/. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Principles of flexible protein-protein docking.

Publication Date: 2008 Jul 24 PMID: 18655061
Authors: Andrusier, N. - Mashiach, E. - Nussinov, R. - Wolfson, H. J.
Journal: Proteins

Treating flexibility in molecular docking is a major challenge in cell biology research. Here we describe the background and the principles of existing flexible protein-protein docking methods, focusing on the algorithms and their rational. We describe how protein flexibility is treated in different stages of the docking process: in the preprocessing stage, rigid and flexible parts are identified and their possible conformations are modeled. This preprocessing provides information for the subsequent docking and refinement stages. In the docking stage, an ensemble of pre-generated conformations or the identified rigid domains may be docked separately. In the refinement stage, small-scale movements of the backbone and side-chains are modeled and the binding orientation is improved by rigid-body adjustments. For clarity of presentation, we divide the different methods into categories. This should allow the reader to focus on the most suitable method for a particular docking problem. Proteins 2008. Published Wiley-Liss, Inc.

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The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state.

Publication Date: 2008 Jul 24 PMID: 18655058
Authors: Almog, O. - Gonzalez, A. - Godin, N. - de Leeuw, M. - Mekel, M. J. - Klein, D. - Braun, S. - Shoham, G. - Walter, R. L.
Journal: Proteins

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Molecular simulations enlighten the binding mode of quercetin to lipoxygenase-3.

Publication Date: 2008 Jul 24 PMID: 18655056
Authors: Fiorucci, S. - Golebiowski, J. - Cabrol-Bass, D. - Antonczak, S.
Journal: Proteins

Inhibition of lipoxygenases (LOXs) by flavonoid compounds is now well documented, but the description of the associated mechanism remains controversial due to a lack of information at the molecular level. For instance, X-ray determination of quercetin/LOX-3 system has led to a structure where the enzyme was cocrystallized with a degradation product of the substrate, which rendered the interpretation of the reported interactions between this flavonoid compound and the enzyme difficult. Molecular modeling simulations can in principle allow obtaining precious insights that could fill this lack of structural information. Thus, in this study, we have investigated various binding modes of quercetin to LOX-3 enzyme in order to understand the first step of the inhibition process, that is the association of the two entities. Molecular dynamics simulations and free energy calculations suggest that quercetin binds the metal center via its 3-hydroxychromone function. Moreover, enzyme/substrate interactions within the cavity impose steric hindrances to quercetin that may activate a direct dioxygen addition on the substrate. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Some recommendations for the practitioner to improve the precision of experimentally determined protein folding rates and phi values.

Publication Date: 2008 Jul 24 PMID: 18655053
Authors: Ruczinski, I. - Plaxco, K. W.
Journal: Proteins

The mechanism by which proteins fold from an initially random conformation into a functional, native structure remains a major unsolved question in molecular biology. Of particular interest to the protein folding community is the structure that the protein adopts in the folding transition state (the highest free energy state on the pathway from unfolded to folded), as that state forms the barrier that defines the folding pathway. Unfortunately, however, unlike those of the initial, unfolded state and the final, folded state of the protein, the structure in the transition state cannot be directly assessed via experiment. Instead, experimentalists infer the structure of the transition state, often by estimating changes in its free energy by measuring the effects of amino acid substitutions on folding and unfolding rates (Phi-value analysis). In this article we show how to obtain more efficient estimates of these important quantities via improved experimental designs, and how to avoid common pitfalls in the analysis of kinetic data during the extraction of these parameters. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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The mechanism of amyloid-fibril formation by stefin B: Temperature and protein concentration dependence of the rates.

Publication Date: 2008 Jul 17 PMID: 18636508
Authors: Skerget, K. - Vilfan, A. - Pompe-Novak, M. - Turk, V. - Waltho, J. P. - Turk, D. - Zerovnik, E.
Journal: Proteins

Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Improving accuracy and efficiency of blind protein-ligand docking by focusing on predicted binding sites.

Publication Date: 2008 Jul 17 PMID: 18636505
Authors: Ghersi, D. - Sanchez, R.
Journal: Proteins

The use of predicted binding sites (binding sites calculated from the protein structure alone) is evaluated here as a tool to focus the docking of small molecule ligands into protein structures, simulating cases where the real binding sites are unknown. The resulting approach consists of a few independent docking runs carried out on small boxes, centered on the predicted binding sites, as opposed to one larger blind docking run that covers the complete protein structure. The focused and blind approaches were compared using a set of 77 known protein-ligand complexes and 19 ligand-free structures. The focused approach is shown to: (1) identify the correct binding site more frequently than blind docking; (2) produce more accurate docking poses for the ligand; (3) require less computational time. Additionally, the results show that very few real binding sites are missed in spite of focusing on only three predicted binding sites per target protein. Overall the results indicate that, by improving the sampling in regions that are likely to correspond to binding sites, the focused docking approach increases accuracy and efficiency of protein ligand docking for those cases where the ligand-binding site is unknown. This is especially relevant in applications such as reverse virtual screening and structure-based functional annotation of proteins. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Geometric constraints for porphyrin binding in helical protein binding sites.

Publication Date: 2008 Jul 17 PMID: 18636480
Authors: Negron, C. - Fufezan, C. - Koder, R. L.
Journal: Proteins

Helical bundles which bind heme and porphyrin cofactors have been popular targets for cofactor-containing de novo protein design. By analyzing a highly nonredundant subset of the protein databank we have determined a rotamer distribution for helical histidines bound to heme cofactors. Analysis of the entire nonredundant database for helical sequence preferences near the ligand histidine demonstrated little preference for amino acid side chain identity, size, or charge. Analysis of the database subdivided by ligand histidine rotamer, however, reveals strong preferences in each case, and computational modeling illuminates the structural basis for some of these findings. The majority of the rotamer distribution matches that predicted by molecular simulation of a single porphyrin-bound histidine residue placed in the center of an all-alanine helix, and the deviations explain two prominent features of natural heme protein binding sites: heme distortion in the case of the cytochromes C in the m166 histidine rotamer, and a highly prevalent glycine residue in the t73 histidine rotamer. These preferences permit derivation of helical consensus sequence templates which predict optimal side chain-cofactor packing interactions for each rotamer. These findings thus promise to guide future design endeavors not only in the creation of higher affinity heme and porphyrin binding sites, but also in the direction of bound cofactor geometry. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Genome-wide enzyme annotation with precision control: Catalytic families (CatFam) databases.

Publication Date: 2008 Jul 17 PMID: 18636476
Authors: Yu, C. - Zavaljevski, N. - Desai, V. - Reifman, J.
Journal: Proteins

In this article, we present a new method termed CatFam (Catalytic Families) to automatically infer the functions of catalytic proteins, which account for 20-40% of all proteins in living organisms and play a critical role in a variety of biological processes. CatFam is a sequence-based method that generates sequence profiles to represent and infer protein catalytic functions. CatFam generates profiles through a stepwise procedure that carefully controls profile quality and employs nonenzymes as negative samples to establish profile-specific thresholds associated with a predefined nominal false-positive rate (FPR) of predictions. The adjustable FPR allows for fine precision control of each profile and enables the generation of profile databases that meet different needs: function annotation with high precision and hypothesis generation with moderate precision but better recall. Multiple tests of CatFam databases (generated with distinct nominal FPRs) against enzyme and nonenzyme datasets show that the method's predictions have consistently high precision and recall. For example, a 1% FPR database predicts protein catalytic functions for a dataset of enzymes and nonenzymes with 98.6% precision and 95.0% recall. Comparisons of CatFam databases against other established profile-based methods for the functional annotation of 13 bacterial genomes indicate that CatFam consistently achieves higher precision and (in most cases) higher recall, and that (on average) CatFam provides 21.9% additional catalytic functions not inferred by the other similarly reliable methods. These results strongly suggest that the proposed method provides a valuable contribution to the automated prediction of protein catalytic functions. The CatFam databases and the database search program are freely available at http://www.bhsai.org/downloads/catfam.tar.gz. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Permeation of water through the KcsA K(+) channel.

Publication Date: 2008 Jul 17 PMID: 18636474
Authors: Furini, S. - Beckstein, O. - Domene, C.
Journal: Proteins

Previous studies have reported that the KcsA potassium channel has an osmotic permeability coefficient of 4.8 x 10(-12) cm(3)/s, giving it a significantly higher osmotic permeability coefficient than that of some membrane channels specialized in water transport. This high osmotic permeability is proposed to occur when the channel is depleted of potassium ions, the presence of which slow down the water permeation process. The atomic structure of the potassium-depleted KcsA channel and the mechanisms of water permeation have not been well characterized so far. Here, all-atom molecular dynamics simulations, in conjunction with an umbrella sampling strategy and a nonequilibrium approach to simulate pressure gradients are employed to illustrate the permeation of water in the absence of ions through the KcsA K(+) channel. Equilibrium molecular dynamics simulations (95 ns combined total length) identified a possible structure of the potassium-depleted KcsA channel, and umbrella sampling calculations (160 ns combined total length) revealed that this structure is not permeable by water molecules moving along the channel axis. The simulation of a pressure gradient across the channel (30 ns combined total length) identified an alternative permeation pathway with a computed osmotic permeability of approximately (2.7 +/- 0.9) x 10(-13) cm(3)/s. Water fluxes along this pathway did not proceed through collective water motions or transitions to vapor state. All of the major results of this study were robust against variations in a wide set of simulation parameters (force field, water model, membrane model, and channel conformation). Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Structure of HP0564 from Helicobacter pylori identifies it as a new transcriptional regulator.

Publication Date: 2008 Jul 11 PMID: 18623065
Authors: Borin, B. N. - Krezel, A. M.
Journal: Proteins

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The crystal structure of the ING5 PHD finger in complex with an H3K4me3 histone peptide.

Publication Date: 2008 Jul 11 PMID: 18623064
Authors: Champagne, K. S. - Saksouk, N. - Pena, P. V. - Johnson, K. - Ullah, M. - Yang, X. J. - Cote, J. - Kutateladze, T. G.
Journal: Proteins

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Identification and characterization of folding inhibitors of hen egg lysozyme: An example of a new paradigm of drug design.

Publication Date: 2008 Jul 11 PMID: 18623063
Authors: Caldarini, M. - Vasile, F. - Provasi, D. - Longhi, R. - Tiana, G. - Broglia, R. A.
Journal: Proteins

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Discovery of sarcosine dimethylglycine methyltransferase from Galdieria sulphuraria.

Publication Date: 2008 Jul 11 PMID: 18623062
Authors: McCoy, J. G. - Bailey, L. J. - Ng, Y. H. - Bingman, C. A. - Wrobel, R. - Weber, A. P. - Fox, B. G. - Phillips, G. N. Jr
Journal: Proteins

An enzyme with sarcosine dimethylglycine methyltransferase (SDMT) activity has been identified in the thermophilic eukaryote, Galdieria sulphuraria. The crystal structure of the enzyme, solved to a resolution of 1.95 A, revealed a fold highly similar to that of mycolic acid synthases. The k(cat) and apparent K(M) values were 64.3 min(-1) and 2.0 mM for sarcosine and 85.6 min(-1) and 2.8 mM for dimethylglycine, respectively. Apparent K(M) values of S-adenosylmethionine were 144 and 150 muM for sarcosine and dimethylglycine, respectively, and the enzyme melting temperature was 61.1 degrees C. Modeling of cofactor binding in the active site based on the structure of methoxy mycolic acid synthase 2 revealed a number of conserved interactions within the active site. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Flexibility and charge asymmetry in the activation loop of Src tyrosine kinases.

Publication Date: 2008 Jul 11 PMID: 18623061
Authors: Banavali, N. K. - Roux, B.
Journal: Proteins

Regulated activity of Src kinases is critical for cell growth. Src kinases can be activated by trans-phosphorylation of a tyrosine located in the central activation loop of the catalytic domain. However, because the required exposure of this tyrosine is not observed in the down-regulated X-ray structures of Src kinases, transient partial opening of the activation loop appears to be necessary for such processes. Umbrella sampling molecular dynamics simulations are used to characterize the free energy landscape of opening of the hydrophilic part of the activation loop in the Src kinase Hck. The loop prefers a partially open conformation where Tyr416 has increased accessibility, but remains partly shielded. An asymmetric distribution of the charged residues in the sequence near Tyr416, which contributes to shielding, is found to be conserved in Src family members. A conformational equilibrium involving exchange of electrostatic interactions between the conserved residues Glu310 and Arg385 or Arg409 affects activation loop opening. A mechanism for access of unphosphorylated Tyr416 into an external catalytic site is suggested based on these observations. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide.

Publication Date: 2008 Jul 10 PMID: 18618712
Authors: Di Fiore, A. - Monti, S. M. - Hilvo, M. - Parkkila, S. - Romano, V. - Scaloni, A. - Pedone, C. - Scozzafava, A. - Supuran, C. T. - De Simone, G.
Journal: Proteins

The cytosolic isoform XIII is a recently discovered member of the human carbonic anhydrase (hCA, EC 4.2.1.1) family. It is selectively expressed among other tissues in the reproductive organs, where it may control pH and ion balance regulation, ensuring thus proper fertilization conditions. The authors report here the X-ray crystallographic structure of this isozyme in the unbound state and in complex with a classical sulfonamide inhibitor, namely acetazolamide. A detailed comparison of the obtained structural data with those already reported for other CA isozymes provides novel insights into the catalytic properties of the members of this protein family. On the basis of the inhibitory properties of acetazolamide against various cytosolic/transmembrane isoforms and the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors are here proposed. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Computational protein design with side-chain conformational entropy.

Publication Date: 2008 Jul 10 PMID: 18618711
Authors: Sciretti, D. - Bruscolini, P. - Pelizzola, A. - Pretti, M. - Jaramillo, A.
Journal: Proteins

Recent advances in modeling protein structures at the atomic level have made it possible to tackle "de novo" computational protein design. Most procedures are based on combinatorial optimization using a scoring function that estimates the folding free energy of a protein sequence on a given main-chain structure. However, the computation of the conformational entropy in the folded state is generally an intractable problem, and its contribution to the free energy is not properly evaluated. In this article, we propose a new automated protein design methodology that incorporates such conformational entropy based on statistical mechanics principles. We define the free energy of a protein sequence by the corresponding partition function over rotamer states. The free energy is written in variational form in a pairwise approximation and minimized using the Belief Propagation algorithm. In this way, a free energy is associated to each amino acid sequence: we use this insight to rescore the results obtained with a standard minimization method, with the energy as the cost function. Then, we set up a design method that directly uses the free energy as a cost function in combination with a stochastic search in the sequence space. We validate the methods on the design of three superficial sites of a small SH3 domain, and then apply them to the complete redesign of 27 proteins. Our results indicate that accounting for entropic contribution in the score function affects the outcome in a highly nontrivial way, and might improve current computational design techniques based on protein stability. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Crystal structure of human phosphomavelonate kinase at 1.8 A resolusion.

Publication Date: 2008 Jul 10 PMID: 18618710
Authors: Chang, Q. - Yan, X. X. - Gu, S. Y. - Liu, J. F. - Liang, D. C.
Journal: Proteins

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Crystal structure of a putative DNA methylase TTHA0409 from Thermus thermophilus HB8.

Publication Date: 2008 Jul 10 PMID: 18618709
Authors: Morita, R. - Ishikawa, H. - Nakagawa, N. - Kuramitsu, S. - Masui, R.
Journal: Proteins

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MADAMM: A multistaged docking with an automated molecular modeling protocol.

Publication Date: 2008 Jul 10 PMID: 18618708
Authors: Cerqueira, N. M. - Bras, N. F. - Fernandes, P. A. - Ramos, M. J.
Journal: Proteins

Dealing with receptor flexibility in docking methodology is still a problem. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. In this paper, we present an automated procedure, called MADAMM, that allows flexibilization of both the receptor and the ligand during a multistaged docking with an automated molecular modeling protocol. We show that the orientation of particular residues at the interface between the protein and the ligand have a crucial influence on the way they interact during the docking process, and the standard docking methodologies failed to predict their correct mode of binding. We present some examples that demonstrate the capabilities of this approach when compared with traditional docking methodologies. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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The structure of the PP2A regulatory subunit B56gamma: The remaining piece of the PP2A jigsaw puzzle.

Publication Date: 2008 Jul 10 PMID: 18618707
Authors: Magnusdottir, A. - Stenmark, P. - Flodin, S. - Nyman, T. - Kotenyova, T. - Graslund, S. - Ogg, D. - Nordlund, P.
Journal: Proteins

The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56gamma. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56gamma and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56gamma first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Local interactions in protein folding determined through an inverse folding model.

Publication Date: 2008 Jul 10 PMID: 18618706
Authors: Bastolla, U. - Porto, M. - Ortiz, A. R.
Journal: Proteins

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Energetics of the loop-to-helix transition leading to the coiled-coil structure of influenza virus hemagglutinin HA2 subunits.

Publication Date: 2008 Jul 10 PMID: 18618705
Authors: Huang, Q. - Korte, T. - Rachakonda, P. S. - Knapp, E. W. - Herrmann, A.
Journal: Proteins

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Prediction of turn types in protein structure by machine-learning classifiers.

Publication Date: 2008 Jul 10 PMID: 18618702
Authors: Meissner, M. - Koch, O. - Klebe, G. - Schneider, G.
Journal: Proteins

We present machine learning approaches for turn prediction from the amino acid sequence. Different turn classes and types were considered based on a novel turn classification scheme. We trained an unsupervised (self-organizing map) and two kernel-based classifiers, namely the support vector machine and a probabilistic neural network. Turn versus non-turn classification was carried out for turn families containing intramolecular hydrogen bonds and three to six residues. Support vector machine classifiers yielded a Matthews correlation coefficient (mcc) of approximately 0.6 and a prediction accuracy of 80%. Probabilistic neural networks were developed for beta-turn type prediction. The method was able to distinguish between five types of beta-turns yielding mcc > 0.5 and at least 80% overall accuracy. We conclude that the proposed new turn classification is distinct and well-defined, and machine learning classifiers are suited for sequence-based turn prediction. Their potential for sequence-based prediction of turn structures is discussed. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Electrostatics-defying interaction between arginine termini as a thermodynamic driving force in protein-protein interaction.

Publication Date: 2008 Jul 10 PMID: 18618701
Authors: Pednekar, D. - Tendulkar, A. - Durani, S.
Journal: Proteins

Apparent electrostatics-defying clustering of arginines attributed as screening effect of solvent is in this study examined as a possible thermodynamic driving force in protein-protein interaction. A dataset of 266 protein dimers is found to have approximately 22% arginines mutually paired and approximately 17% pairs in interaction across interfaces and thus putative "hotspots" of protein-protein interaction. The pairing, uncorrelated with inter or intramolecular context, could be contributing in protein folding as well, and, uncorrelated with solvent access, could be driven by effects that are generic to solvent and protein structures. Mutually stacked at shorter distances but in diverse geometrical modes otherwise, the cations tend to be in gross deficit of hydrogen-bond partners, and contributing electrostatics across protein-protein interface that, on average, is repulsive for protein-protein interaction. Embedded in local environment enriched in polarizable residues, aromatic, aliphatic, and anionic, the arginines may contribute to protein-protein interaction via environmental polarization response to electrostatics of cation clustering, a possible new principle in molecular recognition. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels.

Publication Date: 2008 Jul 10 PMID: 18618700
Authors: Meissner, G. - Pasek, D. A. - Yamaguchi, N. - Ramachandran, S. - Dokholyan, N. V. - Tripathy, A.
Journal: Proteins

The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [(35)S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca(2+) concentrations from <0.01 to 100 muM. At 0.15 muM Ca(2+), [(35)S]CaM bound to RyR2 with decreased affinity and binding enthalpy compared with RyR1. The rates of [(35)S]CaM dissociation from RyR1 increased as the temperature was raised, whereas at 0.15 muM Ca(2+) the rate from RyR2 was little affected. The results suggest major differences in the energetics of CaM binding to and dissociation from RyR1 and RyR2. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Molecular dynamics simulation of the acidic compact state of apomyoglobin from yellowfin tuna.

Publication Date: 2008 Jul 10 PMID: 18618699
Authors: Bismuto, E. - Di Maggio, E. - Pleus, S. - Sikor, M. - Rocker, C. - Nienhaus, G. U. - Lamb, D. C.
Journal: Proteins

A molecular model of the acidic compact state of apomyoglobin (A-state) from yellowfin tuna was obtained using molecular dynamics simulations (MD) by calculating multiple trajectories. To cause partial unfolding within a reasonable amount of CPU time, both an acidic environment (pH 3 and 0.15M NaCl) and a temperature jump to 500 K were needed. Twenty-five acidic structures of apomyoglobin were generated by MD, 10 of them can be clustered by RMSD in an average structure having a common hydrophobic core as was reported for acidic sperm whale apomyoglobin, with shortened helices A,G,E, and H (the helix A appears to be translated along the sequence). Prolonging the MD runs at 500 K did not cause further substantial unfolding, suggesting that the ensemble of generated structures is indicative of a region of the conformational space accessible to the apoprotein at acidic pH corresponding to a local energy minimum. The comparison of experimentally determined values of specific spectroscopic properties of the apomyoglobin in acidic salt conditions with the expected ones on the basis of the MD generated structures shows a reasonable agreement considering the characteristic uncertainties of both experimental and simulation techniques. We used frequency domain fluorometry, acrylamide fluorescence quenching, and fluorescence correlation spectroscopy together with far UV circular dichroism to estimate the helical content, the Stern-Volmer quenching constant and the radius of gyration of the protein. Tuna apomyoglobin is a single tryptophan protein and thus, interpretation of its intrinsic fluorescence is simpler than for other proteins. The high sensitivity of the applied fluorescence techniques enabled experiments to be performed under very dilute conditions, that is, at concentrations of subnanomolar for the FCS measurements and 6 muM for the other fluorescence measurements. As high concentrations of proteins can strongly affect the association equilibrium among partially unfolded states, fluorescence techniques can provide complementary information with respect to other techniques requiring higher sample concentrations, such as NMR. The analysis of exposed hydrophobic regions in each of the MD-generated acidic structures reveals potential candidates involved in the aggregation processes of apomyoglobin in the acidic compact state. Our investigation represents an effective model system for studying amyloid fibril formation found in important diseases that are believed to proceed via aggregation of protein in the molten globule state. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Signaling pathways of PDZ2 domain: A molecular dynamics interaction correlation analysis.

Publication Date: 2008 Jul 10 PMID: 18618698
Authors: Kong, Y. - Karplus, M.
Journal: Proteins

PDZ domains are found in many signaling proteins. One of their functions is to provide scaffolds for forming membrane-associated protein complexes by binding to the carboxyl termini of their partners. PDZ domains are thought also to play a signal transduction role by propagating the information that binding has occurred to remote sites. In this study, a molecular dynamics (MD) simulation-based approach, referred to as an interaction correlation analysis, is applied to the PDZ2 domain to identify the possible signal transduction pathways. A residue correlation matrix is constructed from the interaction energy correlations between all residue pairs obtained from the MD simulations. Two continuous interaction pathways, starting at the ligand binding pocket, are identified by a hierarchical clustering analysis of the residue correlation matrix. One pathway is mainly localized at the N-terminal side of helix alpha1 and the adjacent C-terminus of loop beta1-beta2. The other pathway is perpendicular to the central beta-sheet and extends toward the side of PDZ2 domain opposite to the ligand binding pocket. The results complement previous studies based on multiple sequence analysis, NMR, and MD simulations. Importantly, they reveal the energetic origin of the long-range coupling. The PDZ2 results, as well as the earlier rhodopsin analysis, show that the interaction correlation analysis is a robust approach for determining pathways of intramolecular signal transduction. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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NMR structural studies of the Ste11 SAM domain in the dodecyl phosphocholine micelle.

Publication Date: 2008 Jul 10 PMID: 18618697
Authors: Bhunia, A. - Domadia, P. N. - Mohanram, H. - Bhattacharjya, S.
Journal: Proteins

The sterile alpha-motif (SAM), a relatively small ( approximately 70 amino acids) interaction domain, is found in a variety of proteins involved in cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase from the mitogen activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of full-length protein. In solution, the Ste11 SAM domain exists as a well-folded dimeric structure that is involved in interaction with the cognate SAM domain from an adaptor protein Ste50. In this work, we show that the Ste11 SAM domain has an intrinsic affinity towards the lipid membranes. The solution conformation of the Ste11 SAM determined in perdeuterated DPC micelle, using NMR spectroscopy, is defined by five helices of different lengths connected by a number of loops. In the micelle bound state, the non-polar and aromatic residues of the Ste11 SAM lack a native-like packing and are presumably engaged in interactions with the micelle. Using two different paramagnetic doxyl-lipids; we have mapped out localization of Ste11 SAM residues at the micelle surface. Most of the residues appear to localize at the interfacial region of the micelle. However, a number of non-polar residues from the central region of the domain are found to be located inside the core of the micelle including residues from the helix 4 and a loop between helix 2 and helix 3. Isothermal titration calorimetry studies demonstrate that a facile insertion of the Ste11 SAM into the DPC micelle is primarily driven by a large change in enthalpy, -50 kcal/mol with an apparent equilibrium association constant (K(a)) of 7.86 x 10(6) M(-1). Interestingly, an interfacial mutant L60R of the Ste11 SAM lacking the dimeric structure does not show detectable interactions with the lipid micelle. The micelle-bound structure of the Ste11 SAM domain described in this work may have potential implications in the regulation of MAPK signaling whereby positioning of the Ste11 protein in close proximity to the membrane may facilitate efficient phosphorylation of the Ste11 kinase by the membrane attached upstream Ste20/pak kinase. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme.

Publication Date: 2008 Jul 10 PMID: 18618696
Authors: Montioli, R. - Cellini, B. - Bertoldi, M. - Paiardini, A. - Voltattorni, C. B.
Journal: Proteins

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 muM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Rapid comparison of properties on protein surface.

Publication Date: 2008 Jul 10 PMID: 18618695
Authors: Sael, L. - La, D. - Li, B. - Rustamov, R. - Kihara, D.
Journal: Proteins

The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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The first high pH structure of Escherichia coli aspartate transcarbamoylase.

Publication Date: 2008 Jul 10 PMID: 18618694
Authors: Stieglitz, K. A. - Xia, J. - Kantrowitz, E. R.
Journal: Proteins

The activity and cooperativity of Escherichia coli aspartate transcarbamoylase (ATCase) vary as a function of pH, with a maximum of both parameters at approximately pH 8.3. Here we report the first X-ray structure of unliganded ATCase at pH 8.5, to establish a structural basis for the observed Bohr effect. The overall conformation of the active site at pH 8.5 more closely resembles the active site of the enzyme in the R-state structure than other T-state structures. In the structure of the enzyme at pH 8.5 the 80's loop is closer to its position in R-state structures. A unique electropositive channel, comprised of residues from the 50's region, is observed in this structure, with Arg54 positioned in the center of the channel. The planar angle between the carbamoyl phosphate and aspartate domains of the catalytic chain is more open at pH 8.5 than in ATCase structures determined at lower pH values. The structure of the enzyme at pH 8.5 also exhibits lengthening of a number of interactions in the interface between the catalytic and regulatory chains, whereas a number of interactions between the two catalytic trimers are shortened. These alterations in the interface between the upper and lower trimers may directly shift the allosteric equilibrium and thus the cooperativity of the enzyme. Alterations in the electropositive environment of the active site and alterations in the position of the catalytic chain domains may be responsible for the enhanced activity of the enzyme at pH 8.5. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Solution structure of the GUCT domain from human RNA helicase II/Gubeta reveals the RRM fold, but implausible RNA interactions.

Publication Date: 2008 Jul 9 PMID: 18615715
Authors: Ohnishi, S. - Paakkonen, K. - Koshiba, S. - Tochio, N. - Sato, M. - Kobayashi, N. - Harada, T. - Watanabe, S. - Muto, Y. - Guntert, P. - Tanaka, A. - Kigawa, T. - Yokoyama, S.
Journal: Proteins

Human RNA helicase II/Gualpha (RH-II/Gualpha) and RNA helicase II/Gubeta (RH-II/Gubeta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gualpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gubeta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gubeta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution.

Publication Date: 2008 Jul 9 PMID: 18615714
Authors: Lanza, D. C. - Silva, J. C. - Assmann, E. M. - Quaresma, A. J. - Bressan, G. C. - Torriani, I. L. - Kobarg, J.
Journal: Proteins

The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N-terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X-ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N-terminus. Ab-initio 3D low resolution models of the full-length conformation of the dimeric constructs 6xHis-FEZ1(1-392) and 6xHis-FEZ1(1-227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C-terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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A novel binding pocket of cyclin-dependent kinase 2.

Publication Date: 2008 Jul 9 PMID: 18615713
Authors: Chen, H. - Van Duyne, R. - Zhang, N. - Kashanchi, F. - Zeng, C.
Journal: Proteins

The cyclin-dependent kinase 2 (cdk2) is a serine/threonine protein kinase that plays a key role in the cell cycle control system of all eukaryotic organisms. It has been a much studied drug target for potential anticancer therapy. Most cdk2 inhibitors in clinical development target almost exclusively the catalytic ATP-binding pocket of cdk2. However, several five amino-acid peptide inhibitors that are directed towards a noncatalytic binding pocket of cdk2 are reported here. Upon binding to this new pocket located at the cdk2 and cyclin interface, these peptide inhibitors are found to disrupt the cdk2/cyclin E complex partially and diminish its kinase activity in vitro. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Analysis of electrostatic contributions to the selectivity of interactions between RING-finger domains and ubiquitin-conjugating enzymes.

Publication Date: 2008 Jul 9 PMID: 18615712
Authors: Scheper, J. - Oliva, B. - Villa-Freixa, J. - Thomson, T. M.
Journal: Proteins

The zinc-coordinated protein motifs known as RING-finger domains, present on a class of ubiquitin ligases (E3's), recruit ubiquitin-conjugating enzymes (E2s), tethering them to substrate proteins for covalent modification with ubiquitin. Each RING-finger domain can recruit different E2s, and these interactions are frequently selective, in that certain RING-finger domains associate preferentially with certain E2s. This selectivity acquires particular biological relevance when the recruited E2s exert specialized functions. We have explored the determinants that specify the presence or absence of experimentally detectable interaction between two RING-finger domains, those on RNF11 and RNF103, and two E2s, UBC13, a specialized E2 that catalyzes ubiquitin chain elongation through Lys63 of ubiquitin, and UbcH7, which mediates polyubiquitylation through Lys48. Through the iterative use of computational predictive tools and experimental validations, we have found that these interactions and their selectivity are partly governed by the combinations of electrostatic interactions linking specific residues of the contact interfaces. Our analysis also predicts that the main determinants of selectivity of these interactions reside on the RING-finger domains, rather than on the E2s. The application of some of these rules of interaction selectivity has permitted us to experimentally manipulate the selectivity of interaction of the RING-finger domain-E2 pairs under study. Proteins 2008. (c) 2008 Wiley-Liss, Inc.

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Predicting helix orientation for coiled-coil dimers.

Publication Date: 2008 Aug 15 PMID: 18506779
Authors: Apgar, J. R. - Gutwin, K. N. - Keating, A. E.
Journal: Proteins

The alpha-helical coiled coil is a structurally simple protein oligomerization or interaction motif consisting of two or more alpha helices twisted into a supercoiled bundle. Coiled coils can differ in their stoichiometry, helix orientation, and axial alignment. Because of the near degeneracy of many of these variants, coiled coils pose a challenge to fold recognition methods for structure prediction. Whereas distinctions between some protein folds can be discriminated on the basis of hydrophobic/polar patterning or secondary structure propensities, the sequence differences that encode important details of coiled-coil structure can be subtle. This is emblematic of a larger problem in the field of protein structure and interaction prediction: that of establishing specificity between closely similar structures. We tested the behavior of different computational models on the problem of recognizing the correct orientation--parallel vs. antiparallel--of pairs of alpha helices that can form a dimeric coiled coil. For each of 131 examples of known structure, we constructed a large number of both parallel and antiparallel structural models and used these to assess the ability of five energy functions to recognize the correct fold. We also developed and tested three sequence-based approaches that make use of varying degrees of implicit structural information. The best structural methods performed similarly to the best sequence methods, correctly categorizing approximately 81% of dimers. Steric compatibility with the fold was important for some coiled coils we investigated. For many examples, the correct orientation was determined by smaller energy differences between parallel and antiparallel structures distributed over many residues and energy components. Prediction methods that used structure but incorporated varying approximations and assumptions showed quite different behaviors when used to investigate energetic contributions to orientation preference. Sequence based methods were sensitive to the choice of residue-pair interactions scored.

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Crystal structure of a putative lysostaphin peptidase from Vibrio cholerae.

Publication Date: 2008 Aug 15 PMID: 18498110
Authors: Ragumani, S. - Kumaran, D. - Burley, S. K. - Swaminathan, S.
Journal: Proteins

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The NMR solution structure of the artificial protein M7 matches the computationally designed model.

Publication Date: 2008 Aug 15 PMID: 18498106
Authors: Stordeur, C. - Dalluge, R. - Birkenmeier, O. - Wienk, H. - Rudolph, R. - Lange, C. - Lucke, C.
Journal: Proteins

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Crystal structures of the Mycobacterium tuberculosis secretory antigen alanine dehydrogenase (Rv2780) in apo and ternary complex forms captures "open" and "closed" enzyme conformations.

Publication Date: 2008 Aug 15 PMID: 18491387
Authors: Tripathi, S. M. - Ramachandran, R.
Journal: Proteins

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Crystal structure of QueC from Bacillus subtilis: an enzyme involved in preQ1 biosynthesis.

Publication Date: 2008 Aug 15 PMID: 18491386
Authors: Cicmil, N. - Huang, R. H.
Journal: Proteins

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Crystal structure of B. subtilis YjcG characterizing the YjcG-like group of 2H phosphoesterase superfamily.

Publication Date: 2008 Aug 15 PMID: 18473364
Authors: Li, D. - Liu, C. - Liang, Y. H. - Li, L. F. - Su, X. D.
Journal: Proteins

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Glutathionylation-triggered conformational changes of glutaredoxin Grx1 from the yeast Saccharomyces cerevisiae.

Publication Date: 2008 Aug 15 PMID: 18473363
Authors: Yu, J. - Zhang, N. N. - Yin, P. D. - Cui, P. X. - Zhou, C. Z.
Journal: Proteins

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Crystal structure of a PduO-type ATP:cobalamin adenosyltransferase from Burkholderia thailandensis.

Publication Date: 2008 Aug 15 PMID: 18473361
Authors: Moon, J. H. - Park, A. K. - Jang, E. H. - Kim, H. S. - Chi, Y. M.
Journal: Proteins

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Partial destabilization of native structure by a combination of heat and denaturant facilitates cold denaturation in a hyperthermophile protein.

Publication Date: 2008 Aug PMID: 18452212
Authors: Chandrayan, S. K. - Guptasarma, P.
Journal: Proteins

Cold denaturation is a phenomenon seen in many different proteins. However, there have been no reports so far of its occurrence in hyperthermophile proteins. Here, using a recombinant triosephosphate isomerase (PfuTIM) from the hyperthermophile archaeon, Pyrococcus furiosus, we show that the heating of this protein through the low temperature side of its thermal unfolding transition in the presence of guanidinium hydrochloride (GdmCl) results in the formation of partially-disordered conformational ensembles that retain considerable native-like secondary and tertiary structure. Unlike PfuTIM itself, these thermochemically obtained partially-disordered PfuTIM ensembles display cold denaturation as they are cooled to room temperature. The protein thus shows hysteresis, adopting different structural states in a manner dependent upon the nature of the heating and cooling treatment, rather than upon the initial and final conditions of temperature and GdmCl concentration, indicating that some sort of a kinetic effect influences structure adoption and retention. The structure lost through cooling of partially-disordered PfuTIM is found to be regained through heating. The ability of GdmCl to thus apparently destabilize the highly thermodynamically and kinetically stable structure of PfuTIM (sufficiently, to cause it to display observable cold-denaturation and heat-renaturation transitions, in real-time, with cooling and heating) offers support to current ideas concerning the how hyperthermophile proteins achieve their high kinetic stabilities, and suggests that desolvation-solvation barriers may be responsible for high kinetic stability.

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Crystal structure of the putative carbohydrate recognition domain of human galectin-related protein.

Publication Date: 2008 Aug PMID: 18433051
Authors: Walti, M. A. - Thore, S. - Aebi, M. - Kunzler, M.
Journal: Proteins

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All-atom replica exchange molecular simulation of protein BBL.

Publication Date: 2008 Aug 15 PMID: 18320591
Authors: Zhang, J. - Li, W. - Wang, J. - Qin, M. - Wang, W.
Journal: Proteins

Downhill folding is one of the most important predictions of energy landscape theory. Recently, the Escherichia coli 2-oxoglutarate dehydrogenase PSBD was described as a first example of global downhill folding (Garcia-Mira et al., Science 2002;298:2191), classification that has been later subject of significant controversy. To help resolve this problem, by using intensive all-atom simulation with explicit water model and the replica exchange method, we sample the phase space of protein BBL and depict the free energy landscape. We give an estimate of the free energy barrier height of 1-2 k(B)T, dependent on the way the energy landscape is projected. We also study the conformational distribution of the transition region and find that the three helices generally take the similar positions as that in the native states whereas their spatial orientations show large variability. We further detect the inconsistency between different signals by individually fitting the thermal denaturation curves of five structural features using two-state model, which gives a wide spread melting temperature of 19 K. All of these features are consistent with a picture of folding with very low cooperativities. Compared with the experimental data (Sadqi et al., Nature 2006; 442:317), our results indicate that the Naf-BBL (pH5.3) may have an even lower barrier height and cooperativity.

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Deriving protein dynamical properties from weighted protein contact number.

Publication Date: 2008 Aug 15 PMID: 18300253
Authors: Lin, C. P. - Huang, S. W. - Lai, Y. L. - Yen, S. C. - Shih, C. H. - Lu, C. H. - Huang, C. C. - Hwang, J. K.
Journal: Proteins

It has recently been shown that in proteins the atomic mean-square displacement (or B-factor) can be related to the number of the neighboring atoms (or protein contact number), and that this relationship allows one to compute the B-factor profiles directly from protein contact number. This method, referred to as the protein contact model, is appealing, since it requires neither trajectory integration nor matrix diagonalization. As a result, the protein contact model can be applied to very large proteins and can be implemented as a high-throughput computational tool to compute atomic fluctuations in proteins. Here, we show that this relationship can be further refined to that between the atomic mean-square displacement and the weighted protein contact-number, the weight being the square of the reciprocal distance between the contacting pair. In addition, we show that this relationship can be utilized to compute the cross-correlation of atomic motion (the B-factor is essentially the auto-correlation of atomic motion). For a nonhomologous dataset comprising 972 high-resolution X-ray protein structures (resolution <2.0 A and sequence identity <25%), the mean correlation coefficient between the X-ray and computed B-factors based on the weighted protein contact-number model is 0.61, which is better than those of the original contact-number model (0.51) and other methods. We also show that the computed correlation maps based on the weighted contact-number model are globally similar to those computed through normal model analysis for some selected cases. Our results underscore the relationship between protein dynamics and protein packing. We believe that our method will be useful in the study of the protein structure-dynamics relationship.

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An integrated approach to inferring gene-disease associations in humans.

Publication Date: 2008 Aug 15 PMID: 18300252
Authors: Radivojac, P. - Peng, K. - Clark, W. T. - Peters, B. J. - Mohan, A. - Boyle, S. M. - Mooney, S. D.
Journal: Proteins

One of the most important tasks of modern bioinformatics is the development of computational tools that can be used to understand and treat human disease. To date, a variety of methods have been explored and algorithms for candidate gene prioritization are gaining in their usefulness. Here, we propose an algorithm for detecting gene-disease associations based on the human protein-protein interaction network, known gene-disease associations, protein sequence, and protein functional information at the molecular level. Our method, PhenoPred, is supervised: first, we mapped each gene/protein onto the spaces of disease and functional terms based on distance to all annotated proteins in the protein interaction network. We also encoded sequence, function, physicochemical, and predicted structural properties, such as secondary structure and flexibility. We then trained support vector machines to detect gene-disease associations for a number of terms in Disease Ontology and provided evidence that, despite the noise/incompleteness of experimental data and unfinished ontology of diseases, identification of candidate genes can be successful even when a large number of candidate disease terms are predicted on simultaneously. Availability: www.phenopred.org.

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fRMSDPred: predicting local RMSD between structural fragments using sequence information.

Publication Date: 2008 Aug 15 PMID: 18300251
Authors: Rangwala, H. - Karypis, G.
Journal: Proteins

The effectiveness of comparative modeling approaches for protein structure prediction can be substantially improved by incorporating predicted structural information in the initial sequence-structure alignment. Motivated by the approaches used to align protein structures, this article focuses on developing machine learning approaches for estimating the RMSD value of a pair of protein fragments. These estimated fragment-level RMSD values can be used to construct the alignment, assess the quality of an alignment, and identify high-quality alignment segments. We present algorithms to solve this fragment-level RMSD prediction problem using a supervised learning framework based on support vector regression and classification that incorporates protein profiles, predicted secondary structure, effective information encoding schemes, and novel second-order pairwise exponential kernel functions. Our comprehensive empirical study shows superior results compared with the profile-to-profile scoring schemes. We also show that for protein pairs with low sequence similarity (less than 12% sequence identity) these new local structural features alone or in conjunction with profile-based information lead to alignments that are considerably accurate than those obtained by schemes that use only profile and/or predicted secondary structure information.

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Structural properties of the human respiratory syncytial virus P protein: evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors.

Publication Date: 2008 Aug 15 PMID: 18300250
Authors: Llorente, M. T. - Taylor, I. A. - Lopez-Vinas, E. - Gomez-Puertas, P. - Calder, L. J. - Garcia-Barreno, B. - Melero, J. A.
Journal: Proteins

The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three-dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein.

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Discrimination of near-native structures in protein-protein docking by testing the stability of local minima.

Publication Date: 2008 Aug 15 PMID: 18300245
Authors: Kozakov, D. - Schueler-Furman, O. - Vajda, S.
Journal: Proteins

Fast Fourier transform (FFT) correlation methods of protein-protein docking, combined with the clustering of low energy conformations, can find a number of local minima on the energy surface. For most complexes, the locations of the near-native structures can be constrained to the 30 largest clusters, each surrounding a local minimum. However, no reliable further discrimination can be obtained by energy measures because the differences in the energy levels between the minima are comparable with the errors in the energy evaluation. In fact, no current scoring function accounts for the entropic contributions that relate to the width rather than the depth of the minima. Since structures at narrow minima loose more entropy, some of the nonnative states can be detected by determining whether or not a local minimum is surrounded by a broad region of attraction on the energy surface. The analysis is based on starting Monte Carlo Minimization (MCM) runs from random points around each minimum, and observing whether a certain fraction of trajectories converge to a small region within the cluster. The cluster is considered stable if such a strong attractor exists, has at least 10 convergent trajectories, is relatively close to the original cluster center, and contains a low energy structure. We studied the stability of clusters for enzyme-inhibitor and antibody-antigen complexes in the Protein Docking Benchmark. The analysis yields three main results. First, all clusters that are close to the native structure are stable. Second, restricting considerations to stable clusters eliminates around half of the false positives, that is, solutions that are low in energy but far from the native structure of the complex. Third, dividing the conformational space into clusters and determining the stability of each cluster, the combined approach is less dependent on a priori information than exploring the potential conformational space by Monte Carlo minimizations.

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Toward better refinement of comparative models: predicting loops in inexact environments.

Publication Date: 2008 Aug 15 PMID: 18300241
Authors: Sellers, B. D. - Zhu, K. - Zhao, S. - Friesner, R. A. - Jacobson, M. P.
Journal: Proteins

Achieving atomic-level accuracy in comparative protein models is limited by our ability to refine the initial, homolog-derived model closer to the native state. Despite considerable effort, progress in developing a generalized refinement method has been limited. In contrast, methods have been described that can accurately reconstruct loop conformations in native protein structures. We hypothesize that loop refinement in homology models is much more difficult than loop reconstruction in crystal structures, in part, because side-chain, backbone, and other structural inaccuracies surrounding the loop create a challenging sampling problem; the loop cannot be refined without simultaneously refining adjacent portions. In this work, we single out one sampling issue in an artificial but useful test set and examine how loop refinement accuracy is affected by errors in surrounding side-chains. In 80 high-resolution crystal structures, we first perturbed 6-12 residue loops away from the crystal conformation, and placed all protein side chains in non-native but low energy conformations. Even these relatively small perturbations in the surroundings made the loop prediction problem much more challenging. Using a previously published loop prediction method, median backbone (N-Calpha-C-O) RMSD's for groups of 6, 8, 10, and 12 residue loops are 0.3/0.6/0.4/0.6 A, respectively, on native structures and increase to 1.1/2.2/1.5/2.3 A on the perturbed cases. We then augmented our previous loop prediction method to simultaneously optimize the rotamer states of side chains surrounding the loop. Our results show that this augmented loop prediction method can recover the native state in many perturbed structures where the previous method failed; the median RMSD's for the 6, 8, 10, and 12 residue perturbed loops improve to 0.4/0.8/1.1/1.2 A. Finally, we highlight three comparative models from blind tests, in which our new method predicted loops closer to the native conformation than first modeled using the homolog template, a task generally understood to be difficult. Although many challenges remain in refining full comparative models to high accuracy, this work offers a methodical step toward that goal.

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NMR structure and dynamics of human ephrin-B2 ectodomain: the functionally critical C-D and G-H loops are highly dynamic in solution.

Publication Date: 2008 Aug 15 PMID: 18300229
Authors: Ran, X. - Qin, H. - Liu, J. - Fan, J. S. - Shi, J. - Song, J.
Journal: Proteins

Eph receptors and ephrins constitute the largest family of receptor tyrosine kinases with 15 individual receptors and nine ligands. Its ectodomains represent attractive targets not only for understanding fundamental mechanisms underlying axon guidance, cell migration, segmentation, tumorigenesis, and bone remodeling, but also for drug screening/design to treat cancers, bone diseases and viral infection. So far no NMR study on the ephrin ectodomains is available and as such their properties in solution still remain unknown. In this study, we presented the first NMR structure and dynamics of the human ephrin-B2 ectodomain as well as its interaction with the receptor EphB2. Strikingly, the NMR study reveals a picture different from those previously obtained by X-ray crystallography. Although in solution it still adopts the same Greek key fold, with the central beta-barrel ( approximately 30% of the molecule) highly similar to that in crystal structures, the other regions are highly dynamic and accessible to the bulk solvent. In particular, the functionally critical C-D and G-H loops of the ephrin-B2 ectodomain are highly flexible as reflected by several NMR probes including hydrogen exchange and (15)N backbone relaxation data. Nevertheless, as revealed by ITC and NMR, the ephrin-B2 ectodomain binds to EphB2 with a K(d) of 22.3 nM to form a tight complex in which the tip of the C-D loop and the C-terminus still remain largely flexible. The present results may bear critical implications in understanding the molecular details as well as designing antagonists of therapeutic interest for Eph-ephrin interactions.

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The conserved salt bridge linking two C-terminal beta/alpha units in homodimeric triosephosphate isomerase determines the folding rate of the monomer.

Publication Date: 2008 Aug 15 PMID: 18300228
Authors: Reyes-Lopez, C. A. - Gonzalez-Mondragon, E. - Benitez-Cardoza, C. G. - Chanez-Cardenas, M. E. - Cabrera, N. - Perez-Montfort, R. - Hernandez-Arana, A.
Journal: Proteins

Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (beta/alpha)(8) barrels, has a conserved salt bridge (Arg189-Asp225 in yeast TIM) that connects the two C-terminal beta/alpha segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild-type enzyme. Replacement of Asp225 by Gln caused minor drops in k(cat) and K(M), but the catalytic efficiency (k(cat)/K(M)) was practically unaffected. Temperature-induced unfolding-refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three-fold larger in the mutant than in wild-type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate-limiting step changed from a second-order (at submicromolar concentrations) to a first-order reaction. These results thus indicate that renaturation of yTIM occurs through a uni-bimolecular mechanism in which refolding of the monomer most likely begins at the C-terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state.

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Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase.

Publication Date: 2008 Aug 15 PMID: 18300227
Authors: Angelucci, F. - Miele, A. E. - Boumis, G. - Dimastrogiovanni, D. - Brunori, M. - Bellelli, A.
Journal: Proteins

Thioredoxin glutathione reductase (TGR) is a key flavoenzyme expressed by schistosomes that bridges two detoxification pathways crucial for the parasite survival in the host's organism. In this article we report the crystal structure (at 2.2 A resolution) of TGR from Schistosoma mansoni (SmTGR), deleted in the last two residues. The structure reveals the peculiar architecture of this chimeric enzyme: the small Glutaredoxin (Grx) domain at the N-terminus is joined to the large thioredoxin reductase (TR) one via an extended complementary surface, involving residues not conserved in the Grx superfamily; the TR domain interacts with an identical partner via its C-terminal domain, forming a dimer with a twisted "W" shape. Although lacking the penultimate Selenocysteine residue (Sec), the enzyme is still able to reduce oxidized glutathione. These data update the interpretation of the interdomain communication in TGR enzymes. The possible function of this enzyme in pathogenic parasites is discussed.

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A template-finding algorithm and a comprehensive benchmark for homology modeling of proteins.

Publication Date: 2008 Aug 15 PMID: 18300226
Authors: Vallat, B. K. - Pillardy, J. - Elber, R.
Journal: Proteins

The first step in homology modeling is to identify a template protein for the target sequence. The template structure is used in later phases of the calculation to construct an atomically detailed model for the target. We have built from the Protein Data Bank (PDB) a large-scale learning set that includes tens of millions of pair matches that can be either a true template or a false one. Discriminatory learning (learning from positive and negative examples) is used to train a decision tree. Each branch of the tree is a mathematical programming model. The decision tree is tested on an independent set from PDB entries and on the sequences of CASP7. It provides significant enrichment of true templates (between 50 and 100%) when compared to PSI-BLAST. The model is further verified by building atomically detailed structures for each of the tentative true templates with modeller. The probability that a true match does not yield an acceptable structural model (within 6 A RMSD from the native structure) decays linearly as a function of the TM structural-alignment score.

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Prediction of interaction sites from apo 3D structures when the holo conformation is different.

Publication Date: 2008 Aug 15 PMID: 18300225
Authors: Murga, L. F. - Ondrechen, M. J. - Ringe, D.
Journal: Proteins

The predictability of catalytic and binding sites from apo structures is addressed for proteins that undergo significant conformational change upon binding. Theoretical microscopic titration curves (THEMATICS), an electrostatics-based method for the prediction of functional sites, is performed on a test set of 24 proteins with both apo and holo structures available. For 23 of these 24 proteins (96%), THEMATICS predicts the correct catalytic or binding site for both the apo and holo forms. For only one of the 24 proteins, THEMATICS makes the correct prediction for the holo structure but fails for the apo structure. The metrics used by THEMATICS to identify functional residues generally are larger in absolute value for the functional residues in the holo forms compared to the corresponding residues in the apo forms. However, even in the apo forms, these identifying metrics are still statistically significantly larger for functional residues than for residues not involved in catalysis or binding. This indicates that some of the unusual electrostatic properties of functional residues are preserved in the apo conformation. Evidence is presented that certain residues immediately surrounding the active catalytic and binding residues impart functionally important chemical and electrostatic properties to the active residues. At least parts of these microenvironments exist in the unbound conformations, such that THEMATICS is able to distinguish the functional residues even in the apo structures.

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Mycobacterium tuberculosis isocitrate lyase (MtbIcl): role of divalent cations in modulation of functional and structural properties.

Publication Date: 2008 Aug 15 PMID: 18275086
Authors: Kumar, R. - Bhakuni, V.
Journal: Proteins

Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg(2+) and Mn(2+) ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic alpha/beta barrel core domain of the enzyme and destabilize it. The inhibitors Zn(2+) and Cd(2+) interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl.

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Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom.

Publication Date: 2008 Aug 15 PMID: 18275084
Authors: Marchi-Salvador, D. P. - Correa, L. C. - Magro, A. J. - Oliveira, C. Z. - Soares, A. M. - Fontes, M. R.
Journal: Proteins

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

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Analysis of the residue-residue coevolution network and the functionally important residues in proteins.

Publication Date: 2008 Aug 15 PMID: 18275083
Authors: Lee, B. C. - Park, K. - Kim, D.
Journal: Proteins

It is a common belief that some residues of a protein are more important than others. In some cases, point mutations of some residues make butterfly effect on the protein structure and function, but in other cases they do not. In addition, the residues important for the protein function tend to be not only conserved but also coevolved with other interacting residues in a protein. Motivated by these observations, the authors propose that there is a network composed of the residues, the residue-residue coevolution network (RRCN), where nodes are residues and links are set when the coevolutionary interaction strengths between residues are sufficiently large. The authors build the RRCN for the 44 diverse protein families. The interaction strengths are calculated by using McBASC algorithm. After constructing the RRCN, the authors identify residues that have high degree of connectivity (hub nodes), and residues that play a central role in network flow of information (C(I) nodes). The authors show that these residues are likely to be functionally important residues. Moreover, the C(I) nodes appear to be more relevant to the function than the hub nodes. Unlike other similar methods, the method described in this study is solely based on sequences. Therefore, the method can be applied to the function annotation of a wider range of proteins.

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Induced fit in liver X receptor beta: a molecular dynamics-based investigation.

Publication Date: 2008 Aug 15 PMID: 18275080
Authors: Beautrait, A. - Karaboga, A. S. - Souchet, M. - Maigret, B.
Journal: Proteins

Ligand induced fit phenomenon occurring at the ligand binding domain of the liver X receptor beta (LXRbeta) was investigated by means of molecular dynamics. Reliability of a 4-ns trajectory was tested from two distinct LXRbeta crystal complexes 1PQ6B/GW and 1PQ9B/T09 characterized by an open and a closed state of the pocket, respectively. Crossed complexes 1PQ6B/T09 and 1PQ9B/GW were then submitted to the same molecular dynamic conditions, which were able to recover LXRbeta conformations similar to the original crystallography data. Analysis of "open to closed" and "closed to open" conformational transitions pointed out the dynamic role of critical residues lining the ligand binding pocket involved in the local remodeling upon ligand binding (e.g., Phe271, Phe329, Phe340, Arg319, Glu281). Altogether, the present study indicates that the molecular dynamic protocol is a consistent approach for managing LXRbeta-related induced fit process. This protocol could therefore be used for refining ligand docking solutions of a structure-based design strategy.

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Compact dimension of denatured states of staphylococcal nuclease.

Publication Date: 2008 Aug 15 PMID: 18275079
Authors: Chow, C. Y. - Wu, M. C. - Fang, H. J. - Hu, C. K. - Chen, H. M. - Tsong, T. Y.
Journal: Proteins

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.

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