The visualCMAT: A web-server to select and interpret correlated mutations/co-evolving residues in protein families

Abstract

The visualCMAT web-server was designed to assist experimental research in the fields of protein/enzyme biochemistry, protein engineering, and drug discovery by providing an intuitive and easy-to-use interface to the analysis of correlated mutations/co-evolving residues. Sequence and structural information describing homologous proteins are used to predict correlated substitutions by the Mutual information-based CMAT approach, classify them into spatially close co-evolving pairs, which either form a direct physical contact or interact with the same ligand (e.g. a substrate or a crystallographic water molecule), and long-range correlations, annotate and rank binding sites on the protein surface by the presence of statistically significant co-evolving positions. The results of the visualCMAT are organized for a convenient visual analysis and can be downloaded to a local computer as a content-rich all-in-one PyMol session file with multiple layers of annotation corresponding to bioinformatic, statistical and structural analyses of the predicted co-evolution, or further studied online using the built-in interactive analysis tools. The online interactivity is implemented in HTML5 and therefore neither plugins nor Java are required. The visualCMAT web-server is integrated with the Mustguseal web-server capable of constructing large structure-guided sequence alignments of protein families and superfamilies using all available information about their structures and sequences in public databases. The visualCMAT web-server can be used to understand the relationship between structure and function in proteins, implemented at selecting hotspots and compensatory mutations for rational design and directed evolution experiments to produce novel enzymes with improved properties, and employed at studying the mechanism of selective ligand's binding and allosteric communication between topologically independent sites in protein structures. The web-server is freely available at https://biokinet.belozersky.msu.ru/visualcmat and there are no login requirements.

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

“…To narrow the search in the mutation library and reduce screening efforts, structure information‐guided saturation mutagenesis methods, such as triple code saturation mutagenesis , have been explored to evolve stereoselective and regioselective enzymes with optimized catalytic profiles . In structure‐based saturation mutagenesis methods, the amino acid residues selected for substitution are mostly confined to specific spatial regions in the protein such as an active center or substrate‐binding domain, rather than potential sites outside of these functional regions, potentially involved in distal effects or residue–residue networks that can modulate enzyme functions . This highlights the need for new approaches which expand the selection of mutational hotspots for saturation mutagenesis to the entire protein molecule, while keeping the mutation libraries focused to a comparative small number of residues.…”

“…Here, using the industrially important 926‐residue pullulanase from Bacillus naganoensis as a target protein, we describe a new approach called ‘evolutionary coupling saturation mutagenesis’ (ECSM), to engineer the enzyme guided by coevolution analysis. Although approaches using sequence covariance to target sites for mutagenesis has been previously described , the recent advances in bioinformatic analysis outlined above provide more reliable identification of coevolution due to direct residue contacts, minimizing covariance due to transitive correlations, and enhancing the effectiveness of this strategy. Moreover, in order to validate the current view that coevolution of two residues most often relies on a first mutation at one residue site, reducing fitness which followed, by a second mutation at a contacting site, resulting in increasing fitness, we performed saturation mutagenesis for the residues forming an evolutionary coupled pair in a sequential manner.…”

Acyltransferases as Tools for Polyketide Synthase Engineering

“…Specifically, the information existing in databases [ 191 , 192 ] is incredibly helpful, not only for direct sequence alignments and phylogenetic analysis [ 193 ], but also for the development of advanced bioinformatics tools supporting the AT-targeted PKS engineering [ 194 ]. The application of such tools allows the identification of new PKS pathways [ 195 ] and/or novel types of ATs, the prediction of AT substrate specificity [ 61 , 62 , 158 ], the analysis of amino acid coevolution in protein sequences [ 196 , 197 , 198 ], and even protein interaction surfaces [ 41 , 199 , 200 ]. Recently, the computational online platform ClusterCAD, which facilitates the selection of natural cis -AT PKS parts to design novel chimeric PKSs for the biosynthesis of small PKS-derived compounds, was developed [ 144 , 201 ].…”

CASBench: A Benchmarking Set of Proteins with Annotated Catalytic and Allosteric Sites in Their Structures

“…Co-evolution between positions located in different binding sites at a considerable distance from each other was described (e.g., in the bacterial transcription factors belonging to the LacI family [17]). Spatially proximal co-evolving residue pairs, as well as long-range correlations, can potentially be used to annotate new binding sites and study the molecular mechanisms of allosteric communication [18].…”