Functional restraints on the patterns of amino acid substitutions: application to sequence-structure homology recognition

Amino acid residues that are involved in functional interactions in proteins have strong evolutionary pressure to remain unchanged and consequently their substitution patterns are different from those that are noninteracting. To characterize and quantify the differences between amino acid substitution patterns due to structural restraints and those under functional restraints, we have made a comparative analysis of families of homologous proteins. Residues classified as having the same amino acid type, secondary structure, accessibility, and side-chain hydrogen bonds are shown to be better conserved if they are close to the active site. We have focused on enzyme families for this analysis since they have functional sites that are easily defined by their catalytic residues. We have derived new sets of environment-specific substitution tables, which we term function-dependent environment-specific substitution tables, where amino acid residues are classified according to their distance from the functional sites. The residues that are within a distance of 9 A from the active site have distinct amino acid substitution patterns when compared to the other sites. The function-dependent environment-specific substitution tables have been tested using the sequence-structure homology recognition program FUGUE and the results compared with the recognition performance obtained using the standard environment-specific substitution tables. Significant improvements are obtained in both recognition performance and alignment accuracy using the function-dependent environment-specific substitution tables (P-value = 0.02, according to the Wilcoxon signed rank test for alignment accuracy). The alignments near the active site are greatly improved with pronounced improvements at lower percentage identities (less than 30%).     

A structural dissection of amino acid substitutions in helical transmembrane proteins

The evolution of protein folds is under strong constraints from their surrounding environment. Although folding in water‐soluble proteins is driven primarily by hydrophobic forces, the nature of the forces that determine the folding and stability of transmembrane proteins are still not fully understood. Furthermore, the chemically heterogeneous lipid bilayer has a non‐uniform effect on protein structure. In this article, we attempt to get an insight into the nature of this effect by examining the impact of various types of local structure environment on amino acid substitution, based on alignments of high‐resolution structures of polytopic helical transmembrane proteins combined with sequences of close homologs. Compared to globular proteins, burying amino acid sidechains, especially hydrophilic ones, led to a lower increase in conservation in both the lipid‐water interface region and the hydrocarbon core region. This observation is due to surface residues in HTM proteins especially in the HC region being relatively highly conserved, suggesting higher evolutionary constraints from their specific interactions with the surrounding lipid molecules. Polar and small residues, particularly Pro and Gly, show a noticeable increase in conservation as they are positioned more towards the centre of the membrane, which is consistent with their recognized key roles in structural stability. In addition, the examination of hydrogen bonds in the membrane environment identified some exposed hydrophilic residues being better conserved when not hydrogen‐bonded to other residues, supporting the importance of lipid‐protein sidechain interactions. The conclusions presented in this study highlight the distinct features of substitution matrices that take into account the membrane environment, and their potential role in improving sequence‐structure alignments of transmembrane proteins. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.

The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.     

Discarding functional residues from the substitution table improves predictions of active sites within three-dimensional structures

Substitutions of individual amino acids in proteins may be under very different evolutionary restraints depending on their structural and functional roles. The Environment Specific Substitution Table (ESST) describes the pattern of substitutions in terms of amino acid location within elements of secondary structure, solvent accessibility, and the existence of hydrogen bonds between side chains and neighbouring amino acid residues. Clearly amino acids that have very different local environments in their functional state compared to those in the protein analysed will give rise to inconsistencies in the calculation of amino acid substitution tables. Here, we describe how the calculation of ESSTs can be improved by discarding the functional residues from the calculation of substitution tables. Four categories of functions are examined in this study: protein–protein interactions, protein–nucleic acid interactions, protein–ligand interactions, and catalytic activity of enzymes. Their contributions to residue conservation are measured and investigated. We test our new ESSTs using the program CRESCENDO, designed to predict functional residues by exploiting knowledge of amino acid substitutions, and compare the benchmark results with proteins whose functions have been defined experimentally. The new methodology increases the Z-score by 98% at the active site residues and finds 16% more active sites compared with the old ESST. We also find that discarding amino acids responsible for protein–protein interactions helps in the prediction of those residues although they are not as conserved as the residues of active sites. Our methodology can make the substitution tables better reflect and describe the substitution patterns of amino acids that are under structural restraints only.     

Sequence patterns derived from the automated prediction of functional residues in structurally-aligned homologous protein families

MOTIVATION: Most proteins have evolved to perform specific functions that are dependent on the adoption of well-defined three-dimensional (3D) structures. Specific patterns of conserved residues in amino acid sequences of divergently evolved proteins are frequently observed; these may reflect evolutionary restraints arising both from the need to maintain tertiary structure and the requirement to conserve residues more directly involved in function. Databases of such sequence patterns are valuable in identifying distant homologues, in predicting function and in the study of evolution. RESULTS: A fully automated database of protein sequence patterns, Functional Protein Sequence Pattern Database (FPSPD), has been derived from the analysis of the conserved residues that are predicted to be functional in structurally aligned homologous families in the HOMSTRAD database. Environment-dependent substitution tables, evolutionary trace analysis, solvent accessibility calculations and 3D-structures were used to obtain the FPSPD. The method yielded 3584 patterns that are considered functional and 3049 patterns that are probably functional. FPSPD could be useful for assigning a protein to a homologous superfamily and thereby providing clues about function. AVAILABILITY: FPSPD is available at http://www-cryst.bioc.cam.ac.uk/~fpspd/     

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.     

TM-MOTIF: an alignment viewer to annotate predicted transmembrane helices and conserved motifs in aligned set of sequences

Multiple sequence alignments become biologically meaningful only if conserved and functionally important residues and secondary structural elements preserved can be identified at equivalent positions. This is particularly important for transmembrane proteins like G-protein coupled receptors (GPCRs) with seven transmembrane helices. TM-MOTIF is a software package and an effective alignment viewer to identify and display conserved motifs and amino acid substitutions (AAS) at each position of the aligned set of homologous sequences of GPCRs. The key feature of the package is to display the predicted membrane topology for seven transmembrane helices in seven colours (VIBGYOR colouring scheme) and to map the identified motifs on its respective helices /loop regions. It is an interactive package which provides options to the user to submit query or pre-aligned set of GPCR sequences to align with a reference sequence, like rhodopsin, whose structure has been solved experimentally. It also provides the possibility to identify the nearest homologue from the available inbuilt GPCR or Olfactory Receptor cluster dataset whose association is already known for its receptor type. AVAILABILITY: The database is available for free at mini@ncbs.res.in.     

Protein structural influences in rhodopsin evolution

Incorporating specific structural information can be important for developing a realistic model of evolution for phylogenetic reconstruction of protein-coding genes. We analyzed 62 sequences of vertebrate rhodopsin. The bovine rhodopsin structure was used to label residue sites by surface accessibility, secondary structure, and transmembrane (TM) location. Residue sites with amino acid differences were identified; using maximum parsimony (MP), homoplasious residues were identified. Residues were analyzed for patterns that would indicate correlation of rate with secondary structure, surface accessibility, or position relative to the lipid bilayer. Surface residues, especially those residing in one of the seven TM helices, were significantly correlated with high rates of amino acid substitution. This category of residues, defined solely by protein structural characteristics, potentially defined a class enriched in homoplasious residues. MP analysis using all sites led to a tree with anomalies in the relationships of amphibian, mammalian, bird, and alligator species. Analysis excluding the structurally defined residue class recovered a more accurate phylogeny. A model is presented for including structural influences on rate in phylogenetic inference.     

Modeling amino acid substitution patterns in orthologous and paralogous genes

We study to what degree patterns of amino acid substitution vary between genes using two models of protein-coding gene evolution. The first divides the amino acids into groups, with one substitution rate for pairs of residues in the same group and a second for those in differing groups. Unlike previous applications of this model, the groups themselves are estimated from data by simulated annealing. The second model makes substitution rates a function of the physical and chemical similarity between two residues. Because we model the evolution of coding DNA sequences as opposed to protein sequences, artifacts arising from the differing numbers of nucleotide substitutions required to bring about various amino acid substitutions are avoided. Using 10 alignments of related sequences (five of orthologous genes and five gene families), we do find differences in substitution patterns. We also find that, although patterns of amino acid substitution vary temporally within the history of a gene, variation is not greater in paralogous than in orthologous genes. Improved understanding of such gene-specific variation in substitution patterns may have implications for applications such as sequence alignment and phylogenetic inference.     

Sequence and structure conservation in a protein core

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. Proteins 33:358–366, 1998. © 1998 Wiley-Liss, Inc.     

Estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a Bayesian Monte Carlo approach

The amino acid sequences of proteins provide rich information for inferring distant phylogenetic relationships and for predicting protein functions. Estimating the rate matrix of residue substitutions from amino acid sequences is also important because the rate matrix can be used to develop scoring matrices for sequence alignment. Here we use a continuous time Markov process to model the substitution rates of residues and develop a Bayesian Markov chain Monte Carlo method for rate estimation. We validate our method using simulated artificial protein sequences. Because different local regions such as binding surfaces and the protein interior core experience different selection pressures due to functional or stability constraints, we use our method to estimate the substitution rates of local regions. Our results show that the substitution rates are very different for residues in the buried core and residues on the solvent-exposed surfaces. In addition, the rest of the proteins on the binding surfaces also have very different substitution rates from residues. Based on these findings, we further develop a method for protein function prediction by surface matching using scoring matrices derived from estimated substitution rates for residues located on the binding surfaces. We show with examples that our method is effective in identifying functionally related proteins that have overall low sequence identity, a task known to be very challenging.     

BATMAS30: amino acid substitution matrix for alignment of bacterial transporters

Aligned amino acid sequences of three functionally independent samples of transmembrane (TM) transport proteins have been analyzed. The concept of TM-kernel is proposed as the most probable transmembrane region of a sequence. The average amino acid composition of TM-kernels differs from the published amino acid composition of transmembrane segments. TM-kernels contain more alanines, glycines, and less polar, charged, and aromatic residues in contrast to non-TM-proteins. There are also differences between TM-kernels of bacterial and eukaryotic proteins. We have constructed amino acid substitution matrices for bacterial TM-kernels, named the BATMAS (BActerial Transmembrane MAtrix of Substitutions) series. In TM-kernels, polar and charged residues, as well as proline and tyrosine, are highly conserved, whereas there are more substitutions within the group of hydrophobic residues, in contrast to non-TM-proteins that have fewer, relatively more conserved, hydrophobic residues. These results demonstrate that alignment of transmembrane proteins should be based on at least two amino acid substitution matrices, one for loops (e.g., the BLOSUM series) and one for TM-segments (the BATMAS series), and the choice of the TM-matrix should be different for eukaryotic and bacterial proteins.     

Predicting reliable regions in protein alignments from sequence profiles

For applications such as comparative modelling one major issue is the reliability of sequence alignments. Reliable regions in alignments can be predicted using sub-optimal alignments of the same pair of sequences. Here we show that reliable regions in alignments can also be predicted from multiple sequence profile information alone.Alignments were created for a set of remotely related pairs of proteins using five different test methods. Structural alignments were used to assess the quality of the alignments and the aligned positions were scored using information from the observed frequencies of amino acid residues in sequence profiles pre-generated for each template structure. High-scoring regions of these profile-derived alignment scores were a good predictor of reliably aligned regions.These profile-derived alignment scores are easy to obtain and are applicable to any alignment method. They can be used to detect those regions of alignments that are reliably aligned and to help predict the quality of an alignment. For those residues within secondary structure elements, the regions predicted as reliably aligned agreed with the structural alignments for between 92% and 97.4% of the residues. In loop regions just under 92% of the residues predicted to be reliable agreed with the structural alignments. The percentage of residues predicted as reliable ranged from 32.1% for helix residues to 52.8% for strand residues.This information could also be used to help predict conserved binding sites from sequence alignments. Residues in the template that were identified as binding sites, that aligned to an identical amino acid residue and where the sequence alignment agreed with the structural alignment were in highly conserved, high scoring regions over 80% of the time. This suggests that many binding sites that are present in both target and template sequences are in sequence-conserved regions and that there is the possibility of translating reliability to binding site prediction.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis.

The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain. The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues. Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not. The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues. However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate. These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence. This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics.     

Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues

Two hydrophobic sequences, 24 and 30 residues long, identify the membrane-spanning segments of chemoreceptor Trg from Escherichia coli. As in other related chemoreceptors, these helical sequences are longer than the minimum necessary for an alpha-helix to span the hydrocarbon region of a biological membrane. Thus, the specific positioning of the segments relative to the hydrophobic part of the membrane cannot be deduced from sequence alone. With the aim of defining the positioning for Trg experimentally, we determined accessibility of a hydrophilic sulfhydryl reagent to cysteines introduced at each position within and immediately outside the two hydrophobic sequences. For both sequences, there was a specific region of uniformly low accessibility, bracketed by regions of substantial accessibility. The two low-accessibility regions were each 19 residues long and were in register in the three-dimensional organization of the transmembrane domain deduced from independent data. None of the four hydrophobic-hydrophilic boundaries for these two membrane-embedded sequences occurred at a charged residue. Instead, they were displaced one to seven residues internal to the charged side chains bracketing the extended hydrophobic sequences. Many hydrophobic sequences, known or predicted to be membrane-spanning, are longer than the minimum necessary helical length, but precise membrane boundaries are known for very few. The cysteine-accessibility approach provides an experimental strategy for determining those boundaries that could be widely applicable.     

Distinguishing structural and functional restraints in evolution in order to identify interaction sites

Structural genomics projects are producing many three-dimensional structures of proteins that have been identified only from their gene sequences. It is therefore important to develop computational methods that will predict sites involved in productive intermolecular interactions that might give clues about functions. Techniques based on evolutionary conservation of amino acids have the advantage over physiochemical methods in that they are more general. However, the majority of techniques neither use all available structural and sequence information, nor are able to distinguish between evolutionary restraints that arise from the need to maintain structure and those that arise from function. Three methods to identify evolutionary restraints on protein sequence and structure are described here. The first identifies those residues that have a higher degree of conservation than expected: this is achieved by comparing for each amino acid position the sequence conservation observed in the homologous family of proteins with the degree of conservation predicted on the basis of amino acid type and local environment. The second uses information theory to identify those positions where environment-specific substitution tables make poor predictions of the overall amino acid substitution pattern. The third method identifies those residues that have highly conserved positions when three-dimensional structures of proteins in a homologous family are superposed. The scores derived from these methods are mapped onto the protein three-dimensional structures and contoured, allowing identification clusters of residues with strong evolutionary restraints that are sites of interaction in proteins involved in a variety of functions. Our method differs from other published techniques by making use of structural information to identify restraints that arise from the structure of the protein and differentiating these restraints from others that derive from intermolecular interactions that mediate functions in the whole organism.     

PHAT: a transmembrane-specific substitution matrix. Predicted hydrophobic and transmembrane

MOTIVATION: Database searching algorithms for proteins use scoring matrices based on average protein properties, and thus are dominated by globular proteins. However, since transmembrane regions of a protein are in a distinctly different environment than globular proteins, one would expect generalized substitution matrices to be inappropriate for transmembrane regions. RESULTS: We present the PHAT (predicted hydrophobic and transmembrane) matrix, which significantly outperforms generalized matrices and a previously published transmembrane matrix in searches with transmembrane queries. We conclude that a better matrix can be constructed by using background frequencies characteristic of the twilight zone, where low-scoring true positives have scores indistinguishable from high-scoring false positives, rather than the amino acid frequencies of the database. The PHAT matrix may help improve the accuracy of sequence alignments and evolutionary trees of membrane proteins.     

Simulating DNA coding sequence evolution with EvolveAGene 3

Phylogenetic reconstruction based upon multiple alignments ofmolecular sequences is important to most branches of modernbiology and is central to molecular evolution. Understandingthe historical relationships among macromolecules depends uponcomputer programs that implement a variety of analytical methods.Because it is impossible to know those historical relationshipswith certainty, assessment of the accuracy of methods and theprograms that implement them requires the use of programs thatrealistically simulate the evolution of DNA sequences. EvolveAGene3 is a realistic coding sequence simulation program that separatesmutation from selection and allows the user to set selectionconditions, including variable regions of selection intensitywithin the sequence and variation in intensity of selectionover branches. Variation includes base substitutions, insertions,and deletions. To the best of my knowledge, it is the only programavailable that simulates the evolution of intact coding sequences.Output includes the true tree and true alignments of the resultingcoding sequence and corresponding protein sequences. A log filereports the frequencies of each kind of base substitution, theratio of transition to transversion substitutions, the ratioof indel to base substitution mutations, and the numbers ofsilent and amino acid replacement mutations. The realism ofthe data sets has been assessed by comparing the d_N/d_S ratio,the ratio of transition to transversion substitutions, and theratio of indel to base substitution mutations of the simulateddata sets with those parameters of real data sets from the "goldstandard" BaliBase collection of structural alignments. Resultsshow that the data sets produced by EvolveAGene 3 are very similarto real data sets, and EvolveAGene 3 is therefore a realisticsimulation program that can be used to evaluate a variety ofprograms and methods in molecular evolution.     

FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties.

FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc. cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy.     

Substitution rates in α-helical transmembrane proteins

It has been shown previously that some membrane proteins have a conserved core of amino acid residues. This idea not only serves to orient helices during model building exercises but may also provide insight into the structural role of residues mediating helix-helix interactions. Using experimentally determined high-resolution structures of alpha-helical transmembrane proteins we show that, of the residues within the hydrophobic transmembrane spans, the residues at lipid and subunit interfaces are more evolutionarily variable than those within the lipid-inaccessible core of a polypeptide's transmembrane domain. This supports the idea that helix-helix interactions within the same polypeptide chain and those at the interface between different polypeptide chains may arise in distinct ways. To show this, we use a new method to estimate the substitution rate of an amino acid residue given an alignment and phylogenetic tree of closely related proteins. This method gives better sensitivity in the otherwise-conserved transmembrane domains than a conventional similarity analysis and is relatively insensitive to the sequences used.