Using a residue clash map to functionally characterize protein recombination hybrids

In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features. This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e. electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption). The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries. Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles. Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries. This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.     

Molecular evolution of protein conformational changes revealed by a network of evolutionarily coupled residues

An improved understanding of protein conformational changes has broad implications for elucidating the mechanisms of various biological processes and for the design of protein engineering experiments. Understanding rearrangements of residue interactions is a key component in the challenge of describing structural transitions. Evolutionary properties of protein sequences and structures are extensively studied; however, evolution of protein motions, especially with respect to interaction rearrangements, has yet to be explored. Here, we investigated the relationship between sequence evolution and protein conformational changes and discovered that structural transitions are encoded in amino acid sequences as coevolving residue pairs. Furthermore, we found that highly coevolving residues are clustered in the flexible regions of proteins and facilitate structural transitions by forming and disrupting their interactions cooperatively. Our results provide insight into the evolution of protein conformational changes and help to identify residues important for structural transitions.     

Regional covariation and its application for predicting protein contact patches

Correlated mutation analysis (CMA) is an effective approach for predicting functional and structural residue interactions from multiple sequence alignments (MSAs) of proteins. As nearby residues may also play a role in a given functional interaction, we were interested in seeing whether covarying sites were clustered, and whether this could be used to enhance the predictive power of CMA. A large‐scale search for coevolving regions within protein domains revealed that if two sites in a MSA covary, then neighboring sites in the alignment also typically covary, resulting in clusters of covarying residues. The program PatchD( http://www.uhnres.utoronto.ca/labs/tillier/ ) was developed to measure the covariation between disconnected sequence clusters to reveal patch covariation. Patches that exhibit strong covariation identify multiple residues that are generally nearby in the protein structure, suggesting that the detection of covarying patches can be used in conjunction with traditional CMA approaches to reveal functional interaction partners. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Energetics of charge-charge interactions between residues adjacent in sequence

Statistical analysis of the residue separation between a pair of ionizable side chains within 4 ? of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.     

Identification of functional motions in the adenylate kinase (ADK) protein family by computational hybrid approaches

Based on integrative computational hybrid approaches that combined statistical coupling analysis (SCA), molecular dynamics (MD), and normal mode analysis (NMA), evolutionarily coupled residues involved in functionally relevant motion in the adenylate kinase protein family were identified. The hybrids identified four top-ranking site pairs that belong to a conserved hydrogen bond network that is involved in the enzyme's flexibility. A second group of top-ranking site pairs was identified in critical regions for functional dynamics, such as those related to enzymatic turnover. The high consistency of the results obtained by SCA with NMA (SCA.NMA) and by SCA.MD hybrid analyses suggests that suitable replacement of the matrix of cross-correlation analysis of atomic fluctuations (derived by using NMA) with those based on MD contributes to the identification of such sites by means of a fast computational calculation. The analysis presented here strongly supports the hypothesis that evolutionary forces, such as coevolution at the sequence level, have promoted functional dynamic properties of the adenylate kinase protein family. Finally, these hybrid approaches can be used to identify, at the residue level, protein motion coordination patterns not previously observed, such as in hinge regions.     

Structural and Functional Restraints on the Occurrence of Single Amino Acid Variations in Human Proteins

Human genetic variation is the incarnation of diverse evolutionary history, which reflects both selectively advantageous and selectively neutral change. In this study, we catalogue structural and functional features of proteins that restrain genetic variation leading to single amino acid substitutions. Our variation dataset is divided into three categories: i) Mendelian disease-related variants, ii) neutral polymorphisms and iii) cancer somatic mutations. We characterize structural environments of the amino acid variants by the following properties: i) side-chain solvent accessibility, ii) main-chain secondary structure, and iii) hydrogen bonds from a side chain to a main chain or other side chains. To address functional restraints, amino acid substitutions in proteins are examined to see whether they are located at functionally important sites involved in protein-protein interactions, protein-ligand interactions or catalytic activity of enzymes. We also measure the likelihood of amino acid substitutions and the degree of residue conservation where variants occur. We show that various types of variants are under different degrees of structural and functional restraints, which affect their occurrence in human proteome.     

Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins.     

Hydrogen Bond Strengths in Phosphorylated and Sulfated Amino Acid Residues

Post-translational modification by the addition of an oxoanion functional group, usually a phosphate group and less commonly a sulfate group, leads to diverse structural and functional consequences in protein systems. Building upon previous studies of the phosphoserine residue (pSer), we address the distinct nature of hydrogen bonding interactions in phosphotyrosine (pTyr) and sulfotyrosine (sTyr) residues. We derive partial charges for these modified residues and then study them in the context of molecular dynamics simulation of model tripeptides and sulfated protein complexes, potentials of mean force for interacting residue pairs, and a survey of the interactions of modified residues among experimental protein structures. Overall, our findings show that for pTyr, bidentate interactions with Arg are particularly dominant, as has been previously demonstrated for pSer. sTyr interactions with Arg are significantly weaker, even as compared to the same interactions made by the Glu residue. Our work sheds light on the distinct nature of these modified tyrosine residues, and provides a physical-chemical foundation for future studies with the goal of understanding their roles in systems of biological interest.     

Predicting the functionally distinct residues in the heme,cation, and substrate-binding sites of peroxidase from stress-tolerant mangrove specie, <Emphasis Type="Italic">Avicennia marina</Emphasis>

Recent work was conducted to predict the structure of functionally distinct regions of Avicennia marina peroxidase (AP) by using the structural coordinates of barley grains peroxidase as the template. This enzyme is utilized by all living organisms in many biosynthetic or degradable processes and in defense against oxidative stress. The homology model showed some distinct structural changes in the heme, calcium, and substrate-binding regions. Val53 was found to be an important coordinating residue between distal calcium ion and the distal heme site while Ser176 is coordinated to the proximal histidine through Ala174 and Leu172. Different ionic and hydrogen-bonded interactions were also observed in AP. Analyses of various substrate–enzyme interactions revealed that the substrate-binding pocket is provided by the residues, His41, Phe70, Gly71, Asp138, His139, and Lys176; the later three residues are not conserved in the peroxidase family. We have also performed structural comparison of the A. marina peroxidase with that of two class III salt-sensitive species, peanut and soybean. Four loop regions were found to have largest structural deviation. The overall protein sequence was also analyzed for the presence of probable post-translational modification sites and the functional significance of these sites were outlined.     

A Combinatorial Approach to Detect Coevolved Amino Acid Networks in Protein Families of Variable Divergence

Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.     

Protein Structure Classification Based on Conserved Hydrophobic Residues

Protein folding is frequently guided by local residue interactions that form clusters in the protein core. The interactions between residue clusters serve as potential nucleation sites in the folding process. Evidence postulates that the residue interactions are governed by the hydrophobic propensities that the residues possess. An array of hydrophobicity scales has been developed to determine the hydrophobic propensities of residues under different environmental conditions. In this work, we propose a graph-theory-based data mining framework to extract and isolate protein structural features that sustain invariance in evolutionary-related proteins, through the integrated analysis of five well-known hydrophobicity scales over the 3D structure of proteins. We hypothesize that proteins of the same homology contain conserved hydrophobic residues and exhibit analogous residue interaction patterns in the folded state. The results obtained demonstrate that discriminatory residue interaction patterns shared among proteins of the same family can be employed for both the structural and the functional annotation of proteins. We obtained on the average 90 percent accuracy in protein classification with a significantly small feature vector compared to previous results in the area. This work presents an elaborate study, as well as validation evidence, to illustrate the efficacy of the method and the correctness of results reported.     

Analysis of tryptophan residues in the staphylococcal multidrug transporter QacA reveals long-distance functional associations of residues on opposite sides of the membrane

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein.     

Protein hot spots: the islands of stability

Understanding the structural basis of protein-protein interactions (PPIs) may shed light on the organization and functioning of signal transduction and metabolic networks and may assist in structure-based design of ligands (drugs) targeting protein-protein interfaces. The residues at the bimolecular interface, designated as the hot spots, contribute most of the free binding energy of PPI. To date, there is no conclusive atomistic explanation for the unique functional properties of the hot spots. We hypothesized that backbone compliance may play a role in protein-protein recognition and in the mechanism of binding of small-molecule compounds to protein surfaces. We used a steered molecular dynamics simulation to explore the compliance properties of the backbone of surface-exposed residues in several model proteins: interleukin-2, mouse double minute protein 2 and proliferating cell nuclear antigen. We demonstrated that protein surfaces exhibit distinct patterns in which highly immobile residues form defined clusters ("stability patches") alternating with areas of moderate to high mobility. These "stability patches" tend to localize in functionally important regions involved in protein-protein recognition. We propose a mechanism by which the distinct structural organization of the hot spots may contribute to their role in mediating PPI and facilitating binding of structurally diverse small-molecule compounds to protein surfaces.     

Development of an unbiased statistical method for the analysis of unigenic evolution

Background  

Unigenic evolution is a powerful genetic strategy involving random mutagenesis of a single gene product to delineate functionally important domains of a protein. This method involves selection of variants of the protein which retain function, followed by statistical analysis comparing expected and observed mutation frequencies of each residue. Resultant mutability indices for each residue are averaged across a specified window of codons to identify hypomutable regions of the protein. As originally described, the effect of changes to the length of this averaging window was not fully eludicated. In addition, it was unclear when sufficient functional variants had been examined to conclude that residues conserved in all variants have important functional roles.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ～10% or less of wild-type, while the other has ～45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.     

Detection of Pairwise Residue Proximity by Covariation Analysis for 3D-Structure Prediction of G-Protein-Coupled Receptors

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.     

Estimates of statistical significance for comparison of individual positions in multiple sequence alignments

Background  

Profile-based analysis of multiple sequence alignments (MSA) allows for accurate comparison of protein families. Here, we address the problems of detecting statistically confident dissimilarities between (1) MSA position and a set of predicted residue frequencies, and (2) between two MSA positions. These problems are important for (i) evaluation and optimization of methods predicting residue occurrence at protein positions; (ii) detection of potentially misaligned regions in automatically produced alignments and their further refinement; and (iii) detection of sites that determine functional or structural specificity in two related families.     

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.