We present a new four-body knowledge-based potential for recognizing the native state of proteins from their misfolded states. This potential was extracted from a large set of protein structures determined by X-ray crystallography using BetaMol, a software based on the recent theory of the beta-complex (β-complex) and quasi-triangulation of the Voronoi diagram of spheres. This geometric construct reflects the size difference among atoms in their full Euclidean metric; property not accounted for in a typical 3D Delaunay triangulation. The ability of this potential to identify the native conformation over a large set of decoys was evaluated. Experiments show that this potential outperforms a potential constructed with a classical Delaunay triangulation in decoy discrimination tests. The addition of a statistical hydrogen bond potential to our four-body potential allows a significant improvement in the decoy discrimination, in such a way that we are able to predict successfully the native structure in 90% of cases. Proteins 2013;.© 2013 Wiley Periodicals, Inc.

Coarse-grained Go models have been widely used for studying protein-folding mechanisms. Despite the simplicity of the model, these can reproduce the essential features of the folding process of a protein. However, it is also known that side chains significantly contribute to the folding mechanism. Hence, it is desirable to incorporate the side chain effects into a coarse-grained Go model. In this study, to distinguish the effects of side chain orientation and to understand how these effects contribute to folding mechanisms, we incorporate into a Cα Go model not only heterogeneous contact energies but also geometrical restraints around two Cα atoms in contact with each other. We confirm that the heterogeneity of contact energies governs the folding pathway of a protein and that the geometric constraints attributed to side chains reproduce cooperative transitions in folding.Proteins 2013; © 2013 Wiley Periodicals, Inc.

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5-aminoimidazole ribonucleotide carboxylase and the 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8-Å resolution crystal structure of the 350-kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single-wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species.Proteins 2013; © 2013 Wiley Periodicals, Inc.

We have determined the structure of P2, the self-priming RdRp from cystovirus ϕ12 in two crystal forms (A, B) at resolutions of 1.7 Å and 2.1 Å. Form A contains Mg2+ bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts-mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo.Proteins 2013; © 2013 Wiley Periodicals, Inc.

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5′-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results.Proteins 2013; © 2013 Wiley Periodicals, Inc.

HIV-1 reverse transcriptase is a critical drug target for HIV treatment, and understanding the exact mechanisms of its function and inhibition would significantly accelerate the development of new anti-HIV drugs. It is well known that structure plays a critical role in protein function, but for reverse transcriptase, structural information has proven to be insufficient – despite enormous effort – to explain the mechanism of inhibition and drug resistance of non-nucleoside reverse transcriptase inhibitors. We hypothesize that the missing link is dynamics, information about the motions of the system. However, many of the techniques that give the best information about dynamics, such as solution NMR and molecular dynamics simulations, cannot be easily applied to a protein as large as reverse transcriptase. As an alternative, we combine elastic network modeling with simultaneous hierarchical clustering of structural and dynamic data. We present an extensive survey of the dynamics of reverse transcriptase bound to a variety of ligands and with a number of mutations, revealing a novel mechanism for drug resistance to non-nucleoside reverse transcriptase inhibitors. Hydrophobic core mutations restore active-state motion to multiple functionally significant regions of HIV-1 RT. This model arises out of a combination of structural and dynamic information, rather than exclusively from one or the other. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

We show the accuracy and applicability of our fast algorithmic implementation of a 3-dimensional Poisson-Nernst-Planck (3D PNP) flow model for characterizing different protein channels. Due to its high computational efficiency, our model can predict the full current-voltage characteristics of a channel within minutes, based on the experimental 3D structure of the channel or its computational model structure. Compared with other methods, such as Brownian dynamics, which currently needs a few weeks of the computational time, or even much more demanding molecular dynamics modeling, 3D-PNP is the only available method for a function-based evaluation of the very numerous tentative structural channel models. Flow model tests of our algorithm and its optimal parametrization are provided for five native channels whose experimental structures are available in the protein data bank (PDB) in an open conductive state, and whose experimental current-voltage characteristics have been published. The channels represent very different geometric and structural properties, which makes it the widest test to date of the accuracy of 3D-PNP on real channels. We test whether the channel conductance, rectification and charge selectivity obtained from the flow model, could be sufficiently sensitive to single point mutations, related to unsignificant changes in the channel structure. Our results show that the classical 3D-PNP model, under proper parametrization, is able to achieve a qualitative agreement with experimental data for a majority of the tested characteristics and channels, including channels with narrow and irregular conductivity pores. We propose that although the standard PNP model cannot provide insight into complex physical phenomena due to its intrinsic limitations, its semi-quantitative agreement is achievable for rectification and selectivity at a level sufficient for the bioinformatical purpose with a great advantage of a very short computational time. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Delineation of the relationship between sequence and structure in proteins has proven elusive. Most studies of this problem use alignment methods and other approaches based on the characteristics of individual residues. It is demonstrated herein that the sequence-structure relationship is determined in significant part by global characteristics of sequence organization. Information encoded in complete sequences is required to distinguish proteins in different architectural groups. It is found that the statistically significant differences between sequences encoding different architectures are encoded in a surprisingly small set of low-wave-number sequence periodicities. It would therefore appear that unexpected simplicity in an appropriately defined Fourier space may be an inherent characteristic of the sequences of folded proteins. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

The POLYFIT rigid-body algorithm for automated global pairwise and multiple protein structural alignment is presented. Smith-Waterman local alignment is used to establish a set of seed equivalences that are extended using Needleman-Wunsch dynamic programming techniques. Structural and functional interaction constraints provided by evolution are encoded as one-dimensional residue physical environment strings for alignment of highly structurally overlapped protein pairs. Local structure alignment of more distantly related pairs is carried out using rigid-body conformational matching of 15-residue fragments, with allowance made for less stringent conformational matching of metal-ion and small molecule ligand-contact, disulphide bridge and cis-peptide correspondences. Protein structural plasticity is accommodated through the stepped adjustment of a single empirical distance parameter value in the calculation of the Smith-Waterman dynamic programming matrix. Structural overlap is used both as a measure of similarity and to assess alignment quality. Pairwise alignment accuracy has been bench-marked against that of ten widely used aligners on the Sippl and Wiederstein set of difficult pairwise structure alignment problems, and more extensively against that of Matt, SALIGN and MUSTANG in pairwise and multiple structural alignments of protein domains with low shared sequence identity in the SCOP-ASTRAL 40% compendium. The results demonstrate the advantages of POLYFIT over other aligners in the efficient and robust identification of matching seed residue positions in distantly related protein targets, and in the generation of longer structurally overlapped alignment lengths. Superposition-based application areas include comparative modelling and protein and ligand design. POLYFIT is available on the web-server at http://polyfit.insa-toulouse.fr © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Up to now, efforts to crystallize the cataract-associated P23T mutant of human γD-crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5Å resolution. The protein exhibits essentially the same overall structure as seen for the wild-type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

The subfamily Iα aminotransferases are typically categorized as having narrow specificity towards carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver-related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow-specificity AATases and broad-specificity TATases, as well as AATases with broader-specificity and TATases with narrower-specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this paper, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25-30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C-terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78% and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using 5-fold cross-validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7% and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/~sachen/EFFLUXpredict/Efflux-RBF.php. We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

By far the most studied multidrug resistance protein is P-glycoprotein. Despite recent structural data, key questions about its function remain. P-glycoprotein (P-gp) is flexible and undergoes large conformational changes as part of its function and in this respect details not only of the export cycle but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P-gp. In agreement with previous reports, we found that P-gp undergoes large conformational changes which tended to result in the nucleotide-binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to the available cross-linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart and thus preventing ATP-hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P-gp. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

In this work, we report the ab initio folding of three different extended helical peptides namely 2khk, N36 and C34 through conventional molecular dynamics simulation at room temperature using implicit solvation model. Employing adaptive hydrogen bond specific charge (AHBC) scheme to account for the polarization effect of hydrogen bonds established during the simulation, the effective folding of the three extended helices were observed with best backbone RMSDs in comparison to the experimental structures over the helical region determined to be 1.30 Å for 2khk, 0.73 Å for N36 and 0.72 Å for C34. In this study, 2khk will be used as a benchmark case serving as a means to compare the ability of polarized (AHBC) and non-polarized force field in the folding of an extended helix. Analyses conducted revealed the ability of the AHBC scheme in effectively folding the extended helix by promoting helix growth through the stabilization of backbone hydrogen bonds upon formation during the folding process. Similar observations were also noted when AHBC scheme was employed during the folding of C34 and N36. However, under Amber03 force field, helical structures formed during the folding of 2khk was not accompanied by stabilization thus highlighting the importance of electrostatic polarization in the folding of helical structures. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to a single subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. F43 of shwanavidin forms a hydrophobic lid that is important for biotin binding. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid only results in a modest 20 – 40% improvement in the measured koff. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increases the dissociation half life (t½) from 11 min to 83 min at 20 °C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Antibodies have the capability of binding a wide range of antigens due to the diversity of the six loops constituting the complementarity determining region (CDR). Among the six loops, the H3 loop is the most diverse in structure, length, and sequence identity. Prediction of the three-dimensional structures of antibodies, especially the CDR loops, is an important step in the computational design and engineering of novel antibodies for improved affinity and specificity. Although it has been demonstrated that the conformation of the five non-H3 loops can be accurately predicted by comparing their sequences against databases of canonical loop conformations, no such connection has been established for H3 loops. In this work, we present the results for ab initio structure prediction of the H3 loop using conformational sampling and energy calculations with the program Prime on a dataset of 53 loops ranging in length from 4 to 22 residues. When the prediction is performed in the crystal environment and including symmetry mates, the median backbone root mean square deviation (RMSD) is 0.5 Å to the crystal structure, with 91% of cases having an RMSD of less than 2.0 Å. When the prediction is performed in a noncrystallographic environment, where the scaffold is constructed by swapping the H3 loops between homologous antibodies, 70% of cases have an RMSD below 2.0 Å. These results show promise for ab initio loop predictions applied to modeling of antibodies. © 2012 Wiley Periodicals, Inc.

The conserved ‘stem’ domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies (bnAbs) and a potential vaccine antigen for induction of hetero-subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76-106 of the CD-helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt-LAH residues 76-130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1 and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt-LAH. However, in contrast to the previous study immunization of mice with wt-LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti-viral response. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light-driven proton pump. Here, we report the three-dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein-protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C-terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.

The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. © 2012 Wiley Periodicals, Inc.