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The number of antibiotic resistant pathogens is increasing rapidly, and with this comes a substantial socioeconomic cost that threatens much of the world. To alleviate this problem, we must use antibiotics in a more responsible and informed way, further our understanding of the molecular basis of drug resistance, and design new antibiotics. Here, we focus on a key drug-resistant pathogen, Mycobacterium tuberculosis, and computationally analyze trends in drug-resistant mutations in genes of the proteins embA, embB, embC, and katG, which play essential roles in the action of the first-line drugs ethambutol and isoniazid. We use docking to predict binding modes of isoniazid to katG that agree with suggested binding sites found in our laboratory using cryo-EM. Using mutant stability predictions, we recapitulate the idea that resistance occurs when katG's heme cofactor is destabilized rather than due to a decrease in affinity to isoniazid. Conversely, we have identified resistance mutations that affect the affinity of ethambutol more drastically than the affinity of the natural substrate of embB. With this, we illustrate that we can distinguish between the two types of drug resistance-cofactor destabilization and drug affinity reduction-suggesting potential uses in the prediction of novel drug-resistant mutations., (© 2024 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five β-proteins with known, distinct structures and thermostability (T m ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to T m . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and β-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much., (© 2020 Wiley Periodicals, Inc.)

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n=295) and SDHD (n=63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype-phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma risks suggests differing mechanisms of tumorigenesis in SDH-associated HNPGL and pheochromocytoma.

Mutations in the VHL gene lead to von Hippel-Lindau (VHL) disease, a clinically heterogeneous cancer syndrome. Here, we use software and database tools to understand and predict the phenotypes associated with missense mutations in the VHL gene product, pVHL. The protein product pVHL is known to interact with elongin B, elongin C, and the HIF substrate. By analyzing known and predicted interaction sites and predictions of thermodynamic stability change upon mutation, we generate new hypotheses regarding the molecular etiology of renal cell carcinoma (RCC) and pheochromocytoma (PCC) in VHL disease. We find that the molecular causes of RCC and PCC appear to be decoupled. RCC may arise through two distinct mechanisms: disruption of HIF interactions or binding at the elongin B interface. PCC is triggered by mutations which disrupt interactions at the elongin C binding site. These findings have important implications for VHL disease and for nonfamilial RCC, because most cases of clear cell RCC are linked with VHL inactivation. Additionally, predicting effects of genetic variation will be critical as genetic sequencing accelerates; the analytical strategy presented here may elucidate other systems as further data on genetic variation become available.

Although polar amino acids tend to be found on the surface of proteins due to their hydrophilic nature, their important roles within the core of proteins are now becoming better recognized. It has long been understood that a significant number of mainchain functions will not achieve hydrogen bond satisfaction through the formation of secondary structures; in these circumstances, it is generally buried polar residues that provide hydrogen bond satisfaction. Here, we describe an analysis of the hydrogen-bonding of polar amino acids in a set of structurally aligned protein families. This allows us not only to calculate the conservation of each polar residue but also to assess whether conservation is correlated with the hydrogen-bonding potential of polar sidechains. We show that those polar sidechains whose hydrogen-bonding potential is satisfied tend to be more conserved than their unsatisfied or nonhydrogen-bonded counterparts, particularly when buried. Interestingly, these buried and satisfied polar residues are significantly more conserved than buried hydrophobic residues. Forming hydrogen bonds to mainchain amide atoms also influences conservation, with those satisfied buried polar residues that form two hydrogen bonds to mainchain amides being significantly more conserved than those that form only one or none. These results indicate that buried polar residues whose hydrogen-bonding potential is satisfied are likely to have important roles in maintaining protein structure.

The accurate scoring of rigid-body docking orientations represents one of the major difficulties in protein-protein docking prediction. Other challenges are the development of faster and more efficient sampling methods and the introduction of receptor and ligand flexibility during simulations. Overall, good discrimination of near-native docking poses from the very early stages of rigid-body protein docking is essential step before applying more costly interface refinement to the correct docking solutions. Here we explore a simple approach to scoring of rigid-body docking poses, which has been implemented in a program called pyDock. The scheme is based on Coulombic electrostatics with distance dependent dielectric constant, and implicit desolvation energy with atomic solvation parameters previously adjusted for rigid-body protein-protein docking. This scoring function is not highly dependent on specific geometry of the docking poses and therefore can be used in rigid-body docking sets generated by a variety of methods. We have tested the procedure in a large benchmark set of 80 unbound docking cases. The method is able to detect a near-native solution from 12,000 docking poses and place it within the 100 lowest-energy docking solutions in 56% of the cases, in a completely unrestricted manner and without any other additional information. More specifically, a near-native solution will lie within the top 20 solutions in 37% of the cases. The simplicity of the approach allows for a better understanding of the physical principles behind protein-protein association, and provides a fast tool for the evaluation of large sets of rigid-body docking poses in search of the near-native orientation., ((c) 2007 Wiley-Liss, Inc.)

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster., (Proteins 2006. (c) 2006 Wiley-Liss, Inc.)

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions., (Copyright 2005 Wiley-Liss, Inc.)

The accuracy of model selection from decoy ensembles of protein loop conformations was explored by comparing the performance of the Samudrala-Moult all-atom statistical potential (RAPDF) and the AMBER molecular mechanics force field, including the Generalized Born/surface area solvation model. Large ensembles of consistent loop conformations, represented at atomic detail with idealized geometry, were generated for a large test set of protein loops of 2 to 12 residues long by a novel ab initio method called RAPPER that relies on fine-grained residue-specific phi/psi propensity tables for conformational sampling. Ranking the conformers on the basis of RAPDF scores resulted in selected conformers that had an average global, non-superimposed RMSD for all heavy mainchain atoms ranging from 1.2 A for 4-mers to 2.9 A for 8-mers to 6.2 A for 12-mers. After filtering on the basis of anchor geometry and RAPDF scores, ranking by energy minimization of the AMBER/GBSA potential energy function selected conformers that had global RMSD values of 0.5 A for 4-mers, 2.3 A for 8-mers, and 5.0 A for 12-mers. Minimized fragments had, on average, consistently lower RMSD values (by 0.1 A) than their initial conformations. The importance of the Generalized Born solvation energy term is reflected by the observation that the average RMSD accuracy for all loop lengths was worse when this term is omitted. There are, however, still many cases where the AMBER gas-phase minimization selected conformers of lower RMSD than the AMBER/GBSA minimization. The AMBER/GBSA energy function had better correlation with RMSD to native than the RAPDF. When the ensembles were supplemented with conformations extracted from experimental structures, a dramatic improvement in selection accuracy was observed at longer lengths (average RMSD of 1.3 A for 8-mers) when scoring with the AMBER/GBSA force field. This work provides the basis for a promising hybrid approach of ab initio and knowledge-based methods for loop modeling., (Copyright 2003 Wiley-Liss, Inc.)

We describe a novel method to generate ensembles of conformations of the main-chain atoms [N, C(alpha), C, O, Cbeta] for a sequence of amino acids within the context of a fixed protein framework. Each conformation satisfies fundamental stereo-chemical restraints such as idealized geometry, favorable phi/psi angles, and excluded volume. The ensembles include conformations both near and far from the native structure. Algorithms for effective conformational sampling and constant time overlap detection permit the generation of thousands of distinct conformations in minutes. Unlike previous approaches, our method samples dihedral angles from fine-grained phi/psi state sets, which we demonstrate is superior to exhaustive enumeration from coarse phi/psi sets. Applied to a large set of loop structures, our method samples consistently near-native conformations, averaging 0.4, 1.1, and 2.2 A main-chain root-mean-square deviations for four, eight, and twelve residue long loops, respectively. The ensembles make ideal decoy sets to assess the discriminatory power of a selection method. Using these decoy sets, we conclude that quality of anchor geometry cannot reliably identify near-native conformations, though the selection results are comparable to previous loop prediction methods. In a subsequent study (de Bakker et al.: Proteins 2003;51:21-40), we demonstrate that the AMBER forcefield with the Generalized Born solvation model identifies near-native conformations significantly better than previous methods., (Copyright 2003 Wiley-Liss, Inc.)

Our approach to fold recognition for the fourth critical assessment of techniques for protein structure prediction (CASP4) experiment involved the use of the FUGUE sequence-structure homology recognition program (http://www-cryst.bioc.cam.ac.uk/fugue), followed by model building. We treat models as hypotheses and examine these to determine whether they explain the available data. Our method depends heavily on environment-specific substitution tables derived from our database of structural alignments of homologous proteins (HOMSTRAD, http://www-cryst.bioc.cam.ac.uk/homstrad/). FUGUE uses these tables to incorporate structural information into profiles created from HOMSTRAD alignments that are matched against a profile created for the target from multiple sequence alignment. In addition, environment-specific substitution tables are used throughout the modeling procedure and as part of the model evaluation. Annotation of sequence alignments with JOY, to reflect local structural features, proved valuable, both for modifying hypotheses, and for rejecting predictions when the expected pattern of conservation is not observed. Our stringency in rejecting incorrect predictions led us to submit a relatively small number of models, including only a low number of false positives, resulting in a high average score., (Copyright 2002 Wiley-Liss, Inc.)

We present a fast ab initio method for the prediction of local conformations in proteins. The program, PETRA, selects polypeptide fragments from a computer-generated database (APD) encoding all possible peptide fragments up to twelve amino acids long. Each fragment is defined by a representative set of eight straight phi/psi pairs, obtained iteratively from a trial set by calculating how fragments generated from them represent the protein databank (PDB). Ninety-six percent (96%) of length five fragments in crystal structures, with a resolution better than 1.5 A and less than 25% identity, have a conformer in the database with less than 1 A root-mean-square deviation (rmsd). In order to select segments from APD, PETRA uses a set of simple rule-based filters, thus reducing the number of potential conformations to a manageable total. This reduced set is scored and sorted using rmsd fit to the anchor regions and a knowledge-based energy function dependent on the sequence to be modelled. The best scoring fragments can then be optimized by minimization of contact potentials and rmsd fit to the core model. The quality of the prediction made by PETRA is evaluated by calculating both the differences in rmsd and backbone torsion angles between the final model and the native fragment. The average rmsd ranges from 1.4 A for three residue loops to 3.9 A for eight residue loops., (Copyright 2000 Wiley-Liss, Inc.)

Correct alignment of the sequence of a target protein with those of homologues of known three-dimensional structure is a key step in comparative modeling. Usually an iterative approach that takes account of the local and overall structural features is required. We describe such an approach that exploits databases of structural alignments of homologous proteins (HOMSTRAD, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately homstrad) and protein superfamilies (CAMPASS, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately campass), in which structure-based alignments are analyzed and formatted with the program JOY (http:/(/)www-cryst.bioc.cam.ac.uk/ approximately joy) to reveal conserved local structural features. The databases facilitate the recognition of a family or superfamily, they assist in the selection of useful parent structures, they are helpful in alignment of the target sequences with the parent set, and are useful for deriving relationships that can be used in validating models. In the iterative approach, a model is constructed on the basis of the proposed sequence alignment and this is then reexpressed in the JOY format and realigned with the parent set. This is repeated until the model and sequence alignment is optimized. We examine the case for comparison and use of multiple structures of family members, rather than a single parent structure. We use the targets attempted by our group in CASP3 to assess the value of such procedures.

In order to study structural aspects of sequence conservation in families of homologous proteins, we have analyzed structurally aligned sequences of 585 proteins grouped into 128 homologous families. The conservation of a residue in a family is defined as the average residue similarity in a given position of aligned sequences. The residue similarities were expressed in the form of log-odd substitution tables that take into account the environments of amino acids in three-dimensional structures. The protein core is defined as those residues that have less then 7% solvent accessibility. The density of a protein core is described in terms of atom packing, which is investigated as a criterion for residue substitution and conservation. Although there is no significant correlation between sequence conservation and average atom packing around nonpolar residues such as leucine, valine and isoleucine, a significant correlation is observed for polar residues in the protein core. This may be explained by the hydrogen bonds in which polar residues are involved; the better their protection from water access the more stable should be the structure in that position.

Three-dimensional models of the five functional modules in human protein kinase C alpha (PKC alpha) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinase (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKC alpha is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKC alpha, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKC alpha are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments.

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution. the polypeptide chain of PBGD is folded into three alpha/beta domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity.

Comparison of the three-dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X-ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190-302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports a model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.