Use of sub-family specific residues of protein modules in recognizing sites of functional and structural specialization

Abstract

Functional and regulatory features of a protein in a living system are determined by several factors such as chemical nature and three-dimensional arrangement of residues in the functional site, spatial and temporal expression of the protein in the cell and molecules that interact with the protein. Our understanding of molecular determinants of function is never complete without an understanding of the roles of various residues in the protein. All members of a protein family or a protein domain family share a common structural fold and usually have a conserved general function. However, there can be functional specialisation among the members of individual sub-families within the domain families. This specialisation is a consequence of apparently small structural and sequence differences among the members of divergent sub-families within a domain family. In this project, we have classified members of a protein family into sub-families using a rational basis, and then deduced the residues which are specifically conserved within the individual sub-families. The objective of this work is to study the structural and functional specialised roles that the selectively conserved residues are conferring to their respective sub-families. We have considered two protein domain families as case studies for analysis. The first is the SH2 domain family, the members of which have a function of specifically binding to phospho-Tyr residues in proteins. Chapter 2 describes the partitioning of members of the SH2 domain family in the context of the different domain architectures of proteins into which the SH2 domain is incorporated. Two sub-families were defined consisting of proteins with Src-kinase and Socs (suppressor of cytokine signalling) architecture. Domain-architecture specific residues of each subfamily were identified by comparing multiple sequence alignments of SH2 domain sequences from the individual sub-families. Crystal structures of SH2 domains from the Src-kinase and Socs subfamilies were then inspected to determine the structural and functional roles of the selectively conserved residues. Side-chains of many of the selectively conserved residues from each sub-family were found to be involved in functional interactions either with other residues from the non-SH2 regions of the same protein chain or with residues from the phospho-Tyrosine peptide-ligand. This suggests the roles of selectively conserved residues in assisting domain-domain communication within the protein or conferring sequence specificity towards the pTyr-containing sequences to which the SH2 domains from the two sub-families are specialised to bind. The second protein domain family studied is that of the protein kinases. Chapter 3 describes the comparision between two pre-defined sub-families, cyclin-dependent kinase 2 (cdk2) and protein kinase CK2, of Ser/Thr-kinases belonging to the same hierarchical CMGC group. Cdk2-specific and CK2-specific selectively conserved residues were identified through comparison of the respective multiple sequence alignments and their specialised roles were inferred through survey of literature information, including crystal structures of protein-protein complexes involving Cdk2 and CK2. A number of selectively conserved residues from each sub-family were found to be participating in protein-protein interactions involving the respective protein partners of Cdk2 and CK2. Thus, the approach employed in this project helped us to identify subfamily-specific residues which are sites of subfamily-specific functional and/or structural specialisation, and generate diversity within a protein family. Further, the present work enabled us to propose residues which are likely to play roles in conferring subfamily-specific properties and investigation into their roles could form the basis of future experimental studies.