Identification of hot-spot residues in protein-protein interactions by computational docking

Background  

The study of protein-protein interactions is becoming increasingly important for biotechnological and therapeutic reasons. We can define two major areas therein: the structural prediction of protein-protein binding mode, and the identification of the relevant residues for the interaction (so called 'hot-spots'). These hot-spot residues have high interest since they are considered one of the possible ways of disrupting a protein-protein interaction. Unfortunately, large-scale experimental measurement of residue contribution to the binding energy, based on alanine-scanning experiments, is costly and thus data is fairly limited. Recent computational approaches for hot-spot prediction have been reported, but they usually require the structure of the complex.     

Allelic divergence precedes and promotes gene duplication

One of the striking observations from recent whole-genome comparisons is that changes in the number of specialized genes in existing gene families, as opposed to novel taxon-specific gene families, are responsible for the majority of the difference in genome composition between major taxa. Previous models of duplicate gene evolution focused primarily on the role that neutral processes can play in evolutionary divergence after the duplicates are already fixed in the population. By instead including the entire cycle of duplication and divergence, we show that specialized functions are most likely to evolve through strong selection acting on segregating alleles at a single locus, even before the duplicate arises. We show that the fitness relationships that allow divergent alleles to evolve at a single locus largely overlap with the conditions that allow divergence of previously duplicated genes. Thus, a solution to the paradox of the origin of organismal complexity via the expansion of gene families exists in the form of the deterministic spread of novel duplicates via natural selection.     

An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

Opsin gene duplication and divergence in ray-finned fish

Opsin gene sequences were first reported in the 1980s. The goal of that research was to test the hypothesis that human opsins were members of a single gene family and that variation in human color vision was mediated by mutations in these genes. While the new data supported both hypotheses, the greatest contribution of this work was, arguably, that it provided the data necessary for PCR-based surveys in a diversity of other species. Such studies, and recent whole genome sequencing projects, have uncovered exceptionally large opsin gene repertoires in ray-finned fishes (taxon, Actinopterygii). Guppies and zebrafish, for example, have 10 visual opsin genes each. Here we review the duplication and divergence events that have generated these gene collections. Phylogenetic analyses revealed that large opsin gene repertories in fish have been generated by gene duplication and divergence events that span the age of the ray-finned fishes. Data from whole genome sequencing projects and from large-insert clones show that tandem duplication is the primary mode of opsin gene family expansion in fishes. In some instances gene conversion between tandem duplicates has obscured evolutionary relationships among genes and generated unique key-site haplotypes. We mapped amino acid substitutions at so-called key-sites onto phylogenies and this exposed many examples of convergence. We found that dN/dS values were higher on the branches of our trees that followed gene duplication than on branches that followed speciation events, suggesting that duplication relaxes constraints on opsin sequence evolution. Though the focus of the review is opsin sequence evolution, we also note that there are few clear connections between opsin gene repertoires and variation in spectral environment, morphological traits, or life history traits.     

Evolution of specific protein-protein interaction sites following gene duplication

Gene duplication is a common evolutionary process that leads to the expansion and functional diversification of protein subfamilies. The evolutionary events that cause paralogous proteins to bind different protein ligands (functionally diverged interfaces) are investigated and compared to paralogous proteins that bind the same protein ligand (functionally preserved interfaces). We find that functionally diverged interfaces possess more subfamily-specific residues than functionally preserved interfaces. These subfamily-specific residues are usually partially buried at the interface rim and achieve specific binding through optimized hydrogen bond geometries. In addition to optimized hydrogen bond geometries, side-chain modeling experiments suggest that steric effects are also important for binding specificity. Residues that are completely buried at the interface hub are also less conserved in functionally diverged interfaces than in functionally preserved interfaces. Consistent with this finding, hub residues contribute less to free energy of binding in functionally diverged interfaces than in functionally preserved interfaces. Therefore, we propose that protein binding is a delicate balance between binding affinity that primarily occurs at the interface hub and binding specificity that primarily occurs at the interface rim.     

In silico discovery and experimental validation of new protein-protein interactions

We introduce a framework for predicting novel protein-protein interactions (PPIs), based on Fisher's method for combining probabilities of predictions that are based on different data sources, such as the biomedical literature, protein domain and mRNA expression information. Our method compares favorably to our previous method based on text-mining alone and other methods such as STRING. We evaluated our algorithms through the prediction of experimentally found protein interactions underlying Muscular Dystrophy, Huntington's Disease and Polycystic Kidney Disease, which had not yet been recorded in protein-protein interaction databases. We found a 1.74-fold increase in the mean average prediction precision for dysferlin and a 3.09-fold for huntingtin when compared to STRING. The top 10 of predicted interaction partners of huntingtin were analysed in depth. Five were identified previously, and the other five were new potential interaction partners. The full matrix of human protein pairs and their prediction scores are available for download. Our framework can be extended to predict other types of relationships such as proteins in a complex, pathway or related disease mechanisms.     

Evidence and evolutionary analysis of ancient whole-genome duplication in barley predating the divergence from rice

Background  

Well preserved genomic colinearity among agronomically important grass species such as rice, maize, Sorghum, wheat and barley provides access to whole-genome structure information even in species lacking a reference genome sequence. We investigated footprints of whole-genome duplication (WGD) in barley that shaped the cereal ancestor genome by analyzing shared synteny with rice using a ~2000 gene-based barley genetic map and the rice genome reference sequence.     

Structural and evolutionary characterization of the human sorbitol dehydrogenase gene duplication

We have established that two very closely homologous human sorbitol dehydrogenase sequences lie within 0.5 Mb on Chromosome 15. We have defined the relative orientation of SORD1 and SORD2 genes with respect to both the centromere and each other and established their exact chromosome location. In addition, we have identified polymorphic variants in the locus, which may be useful, in association studies to predict predisposition to clinical problems resulting from decreased conversion of cellular sorbitol to fructose. To define the evolutionary relationship of these human genes, SORD from the marmoset was also sequenced for comparison. Marmoset SORD, which appears to be a single gene in this species, shows significantly less homology with either SORD1 or SORD2 than they do with each other, suggesting that the human homologs represent a recent gene duplication event. A hypothesis is presented to explain the retention of the redundant SORD2 sequence in the human genome. Received: 8 May 1998 / Accepted: 1 August 1998     

Revisit on the evolutionary relationship between alternative splicing and gene duplication

Gene duplications and alternative splicing (AS) isoforms are two widespread types of genetic variations that can facilitate diversification of protein function. A number of studies claimed that after gene duplication, two AS isoforms with differential functions can be 'fixed', respectively, in each of the duplicate copies. This simple 'functional-sharing' hypothesis was recently challenged by Roux and Robinson-Rechavi (2011). Instead, they proposed a more sophisticated hypothesis, invoking that less alternative splicing genes tend to be duplicated more frequently, and single-copy genes are younger than duplicate genes, or the 'duplicability-age' hypothesis for short. In this letter, we show that all these genome-wide analyses of AS isoforms actually did not provide clear-cut evidence to nullify the basic idea of functional-sharing hypothesis. After updating our understanding of genome-wide alternative splicing, duplicability and CNV (copy number variation), we argue that the foundation of the duplicability-age hypothesis remains to be justified carefully. Finally, we suggest that a better approach to resolving this controversy is the correspondence analysis of indels (insertions and deletions) between duplicate genes to the genomic exon-intron structure, which can be used to experimentally test the effect of functional-sharing hypothesis.     

Phylogenomics of the oxidative phosphorylation in fungi reveals extensive gene duplication followed by functional divergence

Background  

Oxidative phosphorylation is central to the energy metabolism of the cell. Due to adaptation to different life-styles and environments, fungal species have shaped their respiratory pathways in the course of evolution. To identify the main mechanisms behind the evolution of respiratory pathways, we conducted a phylogenomics survey of oxidative phosphorylation components in the genomes of sixty fungal species.     

Energetics of quasiequivalence: computational analysis of protein-protein interactions in icosahedral viruses.

Quaternary structure polymorphism found in quasiequivalent virus capsids provides a static framework for studying the dynamics of protein interactions. The same protein subunits are found in different structural environments within these particles, and in some cases, the molecular switching required for the polymorphic quaternary interactions is obvious from high-resolution crystallographic studies. Employing atomic resolution structures, molecular mechanics, and continuum electrostatic methods, we have computed association energies for unique subunit interfaces of three icosahedral viruses, black beetle virus, southern bean virus, and human rhinovirus 14. To quantify the chemical determinants of quasiequivalence, the energetic contributions of individual residues forming quasiequivalent interfaces were calculated and compared. The potential significance of the differences in stabilities at quasiequivalent interfaces was then explored with the combinatorial assembly approach. The analysis shows that the unique association energies computed for each virus serve as a sensitive basis set that may determine distinct intermediates and pathways of virus capsid assembly. The pathways for the quasiequivalent viruses displayed isoenergetic oligomers at specific points, suggesting that these may determine the quaternary structure polymorphism required for the assembly of a quasiequivalent particle.     

A computational tool for identifying minimotifs in protein-protein interactions and improving the accuracy of minimotif predictions

Protein-protein interactions are important to understanding cell functions; however, our theoretical understanding is limited. There is a general discontinuity between the well-accepted physical and chemical forces that drive protein-protein interactions and the large collections of identified protein-protein interactions in various databases. Minimotifs are short functional peptide sequences that provide a basis to bridge this gap in knowledge. However, there is no systematic way to study minimotifs in the context of protein-protein interactions or vice versa. Here we have engineered a set of algorithms that can be used to identify minimotifs in known protein-protein interactions and implemented this for use by scientists in Minimotif Miner. By globally testing these algorithms on verified data and on 100 individual proteins as test cases, we demonstrate the utility of these new computation tools. This tool also can be used to reduce false-positive predictions in the discovery of novel minimotifs. The statistical significance of these algorithms is demonstrated by an ROC analysis (P = 0.001).     

Reporter gene imaging of protein-protein interactions in living subjects

In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.     

Functional divergence of Populus MYB158 and MYB189 gene pair created by whole genome duplication

Whole genome duplication (WGD) providesnew genetic material for genome evolution. After a WGD event, some duplicates are lost, while other duplicates still persist and evolve diverse functions. A particular challenge is to understand how this diversity arises. This study identified two WGD-derived duplicates, MYB158 and MYB189, from Populus tomentosa. Populus MYB158 and MYB189 had expression divergence. Populus tomentosa overexpressing MYB158 or MYB189 had similar phenotypes: creep growth, decreased width of xylem and secondary cell wall thickness. Compared to wild-type, neither myb158 mutant nor myb158 myb189 double mutant showed obvious phenotypic variation in P. tomentosa. Although MYB158 and MYB189 proteins could repress the same structural genes involved in lignin, cellulose, and xylan biosynthesis, the two proteins had their own specific regulatory targets. Populus MYB158 could act as the upstream regulator of secondary cell wall NAC master switch and directly represses the expression of the SND1-B2 gene. Taken together, Populus MYB158 and MYB189 have retained similar functions in negatively regulating secondary cell wall biosynthesis, but have evolved partially distinct functions in direct regulation of NAC master switch, with MYB158 playing a more crucial role. Our findings provide new insights into the evolutionary and functional divergence of WGD-derived duplicate genes.     

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.     

Evidence for short-time divergence and long-time conservation of tissue-specific expression after gene duplication

Gene duplication is one of the main mechanisms by which genomes can acquire novel functions. It has been proposed that the retention of gene duplicates can be associated to processes of tissue expression divergence. These models predict that acquisition of divergent expression patterns should be acquired shortly after the duplication, and that larger divergence in tissue expression would be expected for paralogs, as compared to orthologs of a similar age. Many studies have shown that gene duplicates tend to have divergent expression patterns and that gene family expansions are associated with high levels of tissue specificity. However, the timeframe in which these processes occur have rarely been investigated in detail, particularly in vertebrates, and most analyses do not include direct comparisons of orthologs as a baseline for the expected levels of tissue specificity in absence of duplications. To assess the specific contribution of duplications to expression divergence, we combine here phylogenetic analyses and expression data from human and mouse. In particular, we study differences in spatial expression among human-mouse paralogs, specifically duplicated after the radiation of mammals, and compare them to pairs of orthologs in the same species. Our results show that gene duplication leads to increased levels of tissue specificity and that this tends to occur promptly after the duplication event.     

Prediction of protein-protein interactions by docking methods

Recently, developments have been made in predicting the structure of docked complexes when the coordinates of the components are known. The process generally consists of a stage during which the components are combined rigidly and then a refinement stage. Several rapid new algorithms have been introduced in the rigid docking problem and promising refinement techniques have been developed, based on modified molecular mechanics force fields and empirical measures of desolvation, combined with minimisations that switch on the short-range interactions gradually. There has also been progress in developing a benchmark set of targets for docking and a blind trial, similar to the trials of protein structure prediction, has taken place.