Tolerance to the substitution of buried apolar residues by charged residues in the homologous protein structures

Occurrence and accommodation of charged amino acid residues in proteins that are structurally equivalent to buried non-polar residues in homologues have been investigated. Using a dataset of 1,852 homologous pairs of crystal structures of proteins available at 2A or better resolution, 14,024 examples of apolar residues in the structurally conserved regions replaced by charged residues in homologues have been identified. Out of 2,530 cases of buried apolar residues, 1,677 of the equivalent charged residues in homologues are exposed and the rest of the charged residues are buried. These drastic substitutions are most often observed in homologous protein pairs with low sequence identity (<30%) and in large protein domains (>300 residues). Such buried charged residues in the large proteins are often located in the interface of sub-domains or in the interface of structural repeats, Beyond 7A of residue depth of buried apolar residues, or less than 4% of solvent accessibility, almost all the substituting charged residues are buried. It is also observed that acidic sidechains have higher preference to get buried than the positively charged residues. There is a preference for buried charged residues to get accommodated in the interior by forming hydrogen bonds with another sidechain than the main chain. The sidechains interacting with a buried charged residue are most often located in the structurally conserved regions of the alignment. About 50% of the observations involving hydrogen bond between buried charged sidechain and another sidechain correspond to salt bridges. Among the buried charged residues interacting with the main chain, positively charged sidechains form hydrogen bonds commonly with main chain carbonyls while the negatively charged residues are accommodated by hydrogen bonding with the main chain amides. These carbonyls and amides are usually located in the loops that are structurally variable among homologous proteins.     

The role played by environmental residues on sidechain torsional angles within homologous families of proteins: A new method of sidechain modeling

We investigated the conservation of sidechain conformation for each residue within a homologous family of proteins in the Protein Data Bank (PDB) and performed sidechain modeling using this information. The information was represented by the probability of conserved sidechain torsional angles obtained from many families of proteins, and these were calculated for a pair of residues at topologically equivalent positions as a result of structural alignment. Probabilities were obtained for a pair of same amino acids and for a pair of different amino acids. The correlation between environmental residues and the fluctuation of probability was examined for the pair of same amino acid residues, and the simple probability was calculated for the pair of different amino acids. From the results on the same amino acid pairs, 17 amino acids, except for Ala, Gly, and Pro, were divided into two types: those that were influenced and those that were not influenced by the environmental residues. From results on different amino acid pairs, a replacement between large residues, such as Trp, Phe, and Tyr, was performed assuming conservation of their torsional angles within a homologous family of proteins. We performed sidechain modeling for 11 known proteins from their native and modeled backbones, respectively. With the native backbones, the percentage of the χ₁ angle correct within 30° was found to be 67% and 80% for all and core residues, respectively. With the modeled backbones, the percentage of the correct χ₁ angle was found to be 60% and 72% for all and core residues, respectively. To estimate an upper limit on the accuracy for predicting sidechain conformations, we investigated the probability of conserved sidechain torsional angles for highly similar proteins having > 90% sequence identity and <2.5-Å X-ray resolution. In those proteins, 83% of the sidechain conformations were conserved for the χ₁ angle. Proteins 31:355–369, 1998. © 1998 Wiley-Liss, Inc.     

基于结构比较的蛋白质同源模建系统及其评估Ⅱ侧链的安装

至今,有关蛋白质侧链的同源模建,除了在本体模板上安装侧链和少数限制条件下在同源模板上安装侧链的报道外,系统的研究和实施似乎还未见报道。本软件系统ＰＭＯＤＥＬＩＮＧ采用同源移植和“死端排除“相结合的侧链安装策略,对与模板蛋白相应践基具有相似大小和形状的目标残基采用直接移植的方法。其余铡链则用广义“死端排除定则”安装。经众多蛋白的测试,达到了较好的模建品质。     

基于结构比较的蛋白质同源模建系统及其评估Ⅱ侧链的安装

至今,有关蛋白质侧链的同源模建,除了在本体模板上安装侧链和少数限制条件下在同源模板上安装侧链的报道外,系统的研究和实施似乎还未见报道。本软件系统ＰＭＯＤＥＬＩＮＧ采用同源移植和“死端排除“相结合的侧链安装策略,对与模板蛋白相应践基具有相似大小和形状的目标残基采用直接移植的方法。其余铡链则用广义“死端排除定则”安装。经众多蛋白的测试,达到了较好的模建品质。     

Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling

The three-dimensional structures of 41 homologous proteins (belonging to eight families) were compared by pairwise superposition. A subset of 'core' residues was defined as those whose side chains have less than 7% of their surface exposed to solvent. This subset has significantly higher sequence identity and lower root mean square (RMS) alpha carbon separation than for all topologically equivalent residues in the structure, when members of a protein family are superposed. For such superpositions the relationship between RMS distance and percentage sequence identity of this subset of residues is similar to that for all equivalent residues, although some variation is observed between families of proteins which are predominantly beta sheet and those which are mainly alpha helix. The definition of a structurally more conserved core may be useful in model building proteins from an homologous family. The RMS differences of coordinates of structures of proteins with identical sequences are found to be related to the resolutions of the structures.     

Identification of local conformational similarity in structurally variable regions of homologous proteins using protein blocks

Structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. While the conserved regions often superimpose well, the variable regions appear non superimposable. Differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. One of the kinds of differences between 3-D structures of homologous proteins is rigid body displacement. A glaring example is not well superimposed equivalent regions of homologous proteins corresponding to α-helical conformation with different spatial orientations. In a rigid body superimposition, these regions would appear variable although they may contain local similarity. Also, due to high spatial deviation in the variable region, one-to-one correspondence at the residue level cannot be determined accurately. Another kind of difference is conformational variability and the most common example is topologically equivalent loops of two homologues but with different conformations. In the current study, we present a refined view of the "structurally variable" regions which may contain local similarity obscured in global alignment of homologous protein structures. As structural alphabet is able to describe local structures of proteins precisely through Protein Blocks approach, conformational similarity has been identified in a substantial number of 'variable' regions in a large data set of protein structural alignments; optimal residue-residue equivalences could be achieved on the basis of Protein Blocks which led to improved local alignments. Also, through an example, we have demonstrated how the additional information on local backbone structures through protein blocks can aid in comparative modeling of a loop region. In addition, understanding on sequence-structure relationships can be enhanced through our approach. This has been illustrated through examples where the equivalent regions in homologous protein structures share sequence similarity to varied extent but do not preserve local structure.     

Tertiary structural constraints on protein evolutionary diversity: templates, key residues and structure prediction

The pattern of residue substitution in divergently evolving families of globular proteins is highly variable. At each position in a fold there are constraints on the identities of amino acids from both the three-dimensional structure and the function of the protein. To characterize and quantify the structural constraints, we have made a comparative analysis of families of homologous globular proteins. Residues are classified according to amino acid type, secondary structure, accessibility of the sidechain, and existence of hydrogen bonds from sidechain to other sidechains or peptide carbonyl or amide functions. There are distinct patterns of substitution especially where residues are both solvent inaccessible and hydrogen bonded through their sidechains. The patterns of residue substitution can be used to construct templates or to identify 'key' residues if one or more structures are known. Conversely, analysis of conversation and substitution across a large family of aligned sequences in terms of substitution profiles can allow prediction of tertiary environment or indicate a functional role. Similar analyses can be used to test the validity of putative structures if several homologous sequences are available.     

18th Sir Hans Krebs lecture. Knowledge-based protein modelling and design

A systematic technique for protein modelling that is applicable to the design of drugs, peptide vaccines and novel proteins is described. Our approach is knowledge-based, depending on the structures of homologous or analogous proteins and more generally on a relational data base of protein three-dimensional structures. The procedure simultaneously aligns the known tertiary structures, selects fragments from the structurally conserved regions on the basis of sequence homology, aligns these with the 'average structure' or 'framework', builds on the loops selected from homologous proteins or a wider database, substitutes sidechains and energy minimises the resultant model. Applications to modelling an homologous structure, tissue plasminogen activator on the basis of another serine proteinase, and to modelling an analogous protein, HIV viral proteinase on the basis of aspartic proteinases, are described. The converse problem of ab initio design is also addressed: this involves the selection of an amino acid sequence to give a particular tertiary structure, in this case a symmetrical domain of two Greek-key motifs.     

Modularity and homology: modelling of the type II module family from titin.

We report the homology modelling of the structures of the 162 type II modules from the giant multi-domain protein titin (also known as connectin). The package MODELLER was used and implemented in an automated fashion using four experimentally determined structures as templates. Validation of the models was assessed in terms of divergence from the templates and consensus of the alignments. The homology within the whole family of type II modules as well as with the templates is relatively high (20-35% identity and ca 50% similarity). Comparison between the models of domains for which an NMR structure has been solved and the experimental solution gives an estimate of the quality of the modelling. Our results allow us to distinguish between a set of structurally relevant residues, which are conserved throughout the whole family and buried in the hydrophobic core, from the residues that are conserved and exposed. These latter residues are potentially functionally important. Comparison of exposed conserved patches for modules in different regions of the titin molecule suggests potential interaction surfaces. Our results may be tested directly for those modules whose binding partner is known.     

Conserved positions for ribose recognition: importance of water bridging interactions among ATP,ADP and FAD-protein complexes

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.     

Simulations of the static and dynamic molecular conformations of xyloglucan. The role of the fucosylated sidechain in surface-specific sidechain folding.

The hemicellulosic polysaccharide xyloglucan binds with a strong affinity to cellulosic cell wall microfibrils, the resulting heterogeneous network constituting up to 50% of the dry weight of the cell wall in dicotyledonous plants. To elucidate the molecular details of this interaction, we have performed theoretical potential energy calculations of the static and dynamic equilibrium conformations of xyloglucan using the GEGOP software. In particular, we have evaluated the preferred sidechain conformations of hexa-, octa-, deca- and heptadecasaccharide model fragments of xyloglucan for molecules with a cellulose-like, flat, glucan backbone, and a cellobiose-like, twisted, glucan backbone conformation. For the flat backbone conformation the determination of static equilibrium molecular conformations revealed a tendency for sidechains to fold onto one surface of the backbone, defined here as the H1S face, in the fucosylated region of the polymer. This folding produces a molecule that is sterically accessible on the opposite face of the backbone, the H4S face. Typically, this folding onto the H1S surface is significantly stabilized by favorable interactions between the fucosylated, trisaccharide sidechain and the backbone, with some stabilization from adjacent terminal xylosyl sidechains. In contrast, the trisaccharide sidechain folds onto the H4S face of xyloglucan fragments with a twisted backbone conformation. Preliminary NMR data on nonasaccharide fragments isolated from sycamore suspension-cultured cell walls are consistent with the hypothesis that the twisted conformation of xyloglucan represents the solution form of this molecule. Metropolis Monte Carlo (MMC) simulations were employed to assess sidechain flexibility of the heptadecasaccharide fragments. Simulations performed on the flat, rigid, backbone xyloglucan indicate that the trisaccharide sidechain is less mobile than the terminal xylosyl sidechains. MMC calculations on a fully relaxed molecule revealed a positive correlation between a specific trisaccharide sidechain orientation and the 'flatness' of the backbone glucosyl residues adjacent to this sidechain. These results suggest that the trisaccharide sidechain may play a role in the formation of nucleation sites that initiate the binding of these regions to cellulose. Based on these conformational preferences we suggest the following model for the binding of xyloglucan to cellulose. Nucleation of a binding site is initiated by the fucosylated, trisaccharide sidechain that flattens out an adjacent region of the xyloglucan backbone. Upon contacting a cellulose microfibril this region spreads by step-wise flattening of successive segments of the backbone. Self-association of xyloglucan molecules in solution may be prevented by the low frequency of formation of these nucleation sites and the geometry of the molecules in solution.     

Knowledge based modelling of homologous proteins, Part I: Three-dimensional frameworks derived from the simultaneous superposition of multiple structures

An approach is described for modelling the three-dimensional structure of a protein from the tertiary structures of several homologous proteins that have been determined by X-ray analysis. A method is developed for the simultaneous superposition of several protein molecules and for the calculation of an 'average structure' or 'framework'. Investigation of the convergence properties of this method, in the case of both weighted and unweighted least squares, demonstrates that both give a unique answer and the latter is robust for an homologous family of proteins. Multi-dimensional scaling is used to subgroup of the proteins with respect to structural homology. The framework calculated on the basis of the family of homologous proteins, or of an appropriate subgroup, is used to align fragments of the known protein structures of high sequence homology with the unknown. This alignment provides a basis for model building the tertiary structure. Different techniques for using the framework to model the mainchain of various globins and an immunoglobulin domain in the structurally conserved regions are investigated.     

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety‐six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non‐glycyl residues. Reasons for this observation were investigated by in‐silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C^β atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In‐silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C^β atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.     

Conformational properties of the pentapeptide fragment [32-36] of the thymic hormone thymopoietin

The conformational energy for the pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is calculated using empirical potential functions. Calculation of the local interactions for each independent residue gives a local energy term for which the probabilities as a function of phi, psi are plotted on Ramachandran-type maps. The interaction energy between residues is calculated only for these points in the maps with maximum probability. The most probable conformation for TP5 is found to have an extended backbone arrangement having the Arg and Tyr sidechains folded over the backbone. 13C n.m.r. spin lattice relaxation time measurements show no increase in T1 of the alpha-carbons at the first and terminal amino acids. The increase in T1 along the sidechain as found for Lys does not occur for Arg and Tyr. These signs of reduced mobility are consistent with a set of folded conformations in which the Arg and Tyr sidechains have hindered internal rotations. The vicinal NH-C alpha H couplings agree well with those calculated for the most probable conformer. This is not so for the C alpha H-C beta H couplings. These data are consistent with previous n.m.r. and structure activity studies.     

Buried water in homologous serine proteases.

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.     

On the evolutionary conservation of hydrogen bonds made by buried polar amino acids: the hidden joists,braces and trusses of protein architecture

Background  

The hydrogen bond patterns between mainchain atoms in protein structures not only give rise to regular secondary structures but also satisfy mainchain hydrogen bond potential. However, not all mainchain atoms can be satisfied through hydrogen bond interactions that arise in regular secondary structures; in some locations sidechain-to-mainchain hydrogen bonds are required to provide polar group satisfaction. Buried polar residues that are hydrogen-bonded to mainchain amide atoms tend to be highly conserved within protein families, confirming that mainchain architecture is a critical restraint on the evolution of proteins. We have investigated the stabilizing roles of buried polar sidechains on the backbones of protein structures by performing an analysis of solvent inaccessible residues that are entirely conserved within protein families and superfamilies and hydrogen bonded to an equivalent mainchain atom in each family member.     

Autofix for backward-fit sidechains: using MolProbity and real-space refinement to put misfits in their place

Misfit sidechains in protein crystal structures are a stumbling block in using those structures to direct further scientific inference. Problems due to surface disorder and poor electron density are very difficult to address, but a large class of systematic errors are quite common even in well-ordered regions, resulting in sidechains fit backwards into local density in predictable ways. The MolProbity web site is effective at diagnosing such errors, and can perform reliable automated correction of a few special cases such as 180° flips of Asn or Gln sidechain amides, using all-atom contacts and H-bond networks. However, most at-risk residues involve tetrahedral geometry, and their valid correction requires rigorous evaluation of sidechain movement and sometimes backbone shift. The current work extends the benefits of robust automated correction to more sidechain types. The Autofix method identifies candidate systematic, flipped-over errors in Leu, Thr, Val, and Arg using MolProbity quality statistics, proposes a corrected position using real-space refinement with rotamer selection in Coot, and accepts or rejects the correction based on improvement in MolProbity criteria and on χ angle change. Criteria are chosen conservatively, after examining many individual results, to ensure valid correction. To test this method, Autofix was run and analyzed for 945 representative PDB files and on the 50S ribosomal subunit of file 1YHQ. Over 40% of Leu, Val, and Thr outliers and 15% of Arg outliers were successfully corrected, resulting in a total of 3,679 corrected sidechains, or 4 per structure on average. Summary Sentences: A common class of misfit sidechains in protein crystal structures is due to systematic errors that place the sidechain backwards into the local electron density. A fully automated method called “Autofix” identifies such errors for Leu, Val, Thr, and Arg and corrects over one third of them, using MolProbity validation criteria and Coot real-space refinement of rotamers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of hydrophobic core packing on sidechain dynamics

The effect of hydrophobic core packing on sidechain dynamics was analyzed by comparing the dynamics of wild-type (WT) ubiquitin to those of a variant which has seven core mutations. This variant, 1D7, was designed to resemble WT by having a well-packed core of similar volume, and we find that its overall level of dynamics is only subtly different from WT. However, the mutations caused a redistribution in the positions of core residues that are dynamic. This correlates with the tendency of these residues to populate unfavorable rotamers, suggesting that strain from poor sidechain conformations may promote increased flexibility as a mechanism to relieve unfavorable steric interactions. The results demonstrate that even when core volume is conserved, different packing arrangements in mutants can alter dynamic behavior.     

Correlation between local structural dynamics of proteins inferred from NMR ensembles and evolutionary dynamics of homologues of known structure

Conformational changes in proteins are extremely important for their biochemical functions. Correlation between inherent conformational variations in a protein and conformational differences in its homologues of known structure is still unclear. In this study, we have used a structural alphabet called Protein Blocks (PBs). PBs are used to perform abstraction of protein 3-D structures into a 1-D strings of 16 alphabets (a–p) based on dihedral angles of overlapping pentapeptides. We have analyzed the variations in local conformations in terms of PBs represented in the ensembles of 801 protein structures determined using NMR spectroscopy. In the analysis of concatenated data over all the residues in all the NMR ensembles, we observe that the overall nature of inherent local structural variations in NMR ensembles is similar to the nature of local structural differences in homologous proteins with a high correlation coefficient of .94. High correlation at the alignment positions corresponding to helical and β-sheet regions is only expected. However, the correlation coefficient by considering only the loop regions is also quite high (.91). Surprisingly, segregated position-wise analysis shows that this high correlation does not hold true to loop regions at the structurally equivalent positions in NMR ensembles and their homologues of known structure. This suggests that the general nature of local structural changes is unique; however most of the local structural variations in loop regions of NMR ensembles do not correlate to their local structural differences at structurally equivalent positions in homologues.     

Comparative model-building of the mammalian serine proteases

Proteins have been classified into families based upon sequence homology. An accurate, systematic comparative model-building procedure for a homologous family of proteins would be very valuable scientifically. This paper presents such a procedure and applies it to the mammalian serine proteases, which are ubiquitous and involved in many important biological functions. Eleven proteins of this family are considered here, including a variety of blood serum, intestinal and pancreatic proteins as well as a closely related bacterial enzyme.The modeling method capitalizes upon the availability of three experimentally determined structures for mammalian serine proteases. These structures show that the molecule is divided into structurally conserved regions, which contain the strong sequence homology, and structurally variable regions, which include all the additions and deletions. We show that by applying this structural distinction to new sequences, erroneous alignments of the sequences are greatly minimized.For each aligned new sequence, the structurally conserved regions can be constructed from any of the known structures. In examining the variable regions, we have found that a variable region that has the same length and residue character in two different known structures usually has the same conformation in both. Thus, when the eight structurally unknown proteins are modeled, most of the variable regions can be constructed directly from the known structures. A minority of the variable regions require more sophisticated analysis to evaluate the relative merits of a small number of possible conformations. Only a very few are so different that modeling by homology is entirely ruled out. We demonstrate, therefore, that by this modeling procedure, the maximum of each of these mammalian serine proteases is constructed directly from the experimentally determined structures and the necessity to build from intuition or from energy considerations is greatly reduced.