Zebra2 is a highly automated web-tool to search for subfamily-specific and conserved positions (i.e. the determinants of functional diversity as well as the key catalytic and structural residues) in protein superfamilies. The bioinformatic analysis is facilitated by Mustguseal—a companion web-server to automatically collect and superimpose a large representative set of functionally diverse homologs with high structure similarity but low sequence identity to the selected query protein. The results are automatically prioritized and provided at four information levels to facilitate the knowledge-driven expert selection of the most promising positions on-line: as a sequence similarity network; interfaces to sequence-based and 3D-structure-based analysis of conservation and variability; and accompanied by the detailed annotation of proteins accumulated from the integrated databases with links to the external resources. The integration of Zebra2 and Mustguseal web-tools provides the first of its kind out-of-the-box open-access solution to conduct a systematic analysis of evolutionarily related proteins implementing different functions within a shared 3D-structure of the superfamily, determine common and specific patterns of function-associated local structural elements, assist to select hot-spots for rational design and to prepare focused libraries for directed evolution. The web-servers are free and open to all users at https://biokinet.belozersky.msu.ru/zebra2, no login required.

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.

Neuraminidase A (NanA) of the pathogen Streptococcus pneumoniae cleaves receptors of the human respiratory epithelial surface during bacterial colonization. The full-size structure of NanA that contains one lectin and one catalytic domain within a single polypeptide chain remains unresolved. Both domains are crucial for the microorganism's virulence and considered as promising antimicrobial targets. Methods of bioinformatics and molecular dynamics have been implemented to model NanA's structure and study interaction between the lectin and catalytic domains in three neuraminidases NanA, NanB, and NanC from Streptococcus pneumoniae. A significant difference in spatial organization of these homologous enzymes has been revealed. The lectin and catalytic domains of NanB and NanC form rigid globules stabilized by multiple interdomain interactions, whereas in NanA, the two domains are separated by a 16 amino acids long flexible linker - a characteristic of proteins that require conformational flexibility for their functioning. The biological role of this structural adaptation of NanA as a key virulence enzyme is discussed.

Disulfide bonds play a significant role in protein stability, function or regulation but are poorly conserved among evolutionarily related proteins. The Yosshi can help to understand the role of S–S bonds by comparing sequences and structures of homologs with diverse properties and different disulfide connectivity patterns within a common structural fold of a superfamily, and assist to select the most promising hot-spots to improve stability of proteins/enzymes or modulate their functions by introducing naturally occurring crosslinks. The bioinformatic analysis is supported by the integrated Mustguseal web-server to construct large structure-guided sequence alignments of functionally diverse protein families that can include thousands of proteins based on all available information in public databases. The Yosshi+Mustguseal is a new integrated web-tool for a systematic homology-driven analysis and engineering of S–S bonds that facilitates a broader interpretation of disulfides not just as a factor of structural stability, but rather as a mechanism to implement functional diversity within a superfamily. The results can be downloaded as a content-rich PyMol session file or further studied online using the HTML5-based interactive analysis tools. Both web-servers are free and open to all users at https://biokinet.belozersky.msu.ru/yosshi and there is no login requirement.

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