Selectivity and structure of chimeric loop swaps in SH2 domains

SH2 (Src Homology 2) domains are protein domains that bind to phosphorylated tyrosine residues within cell signaling cascades. They have been found to play a role in certain cancers and immunological disorders. Despite their importance in cell signaling and medical relevance, the structural basis of the various selectivity classes of SH2 domains is only partially understood. Previous research found that the EF and BG loops of the domains contribute to forming the peptide binding pocket, and thus impact their selectivity. To further understand the role of these loops in selectivity, we engineered chimeric SH2 domains by swapping the EF and BG loops from other SH2 domains into the backbone of c-Src SH2. Methods of fluorescence polarization, Ki binding assays, computer modeling, and X-ray crystallography were used to test if the loops can alter selectivity and structure. We found that the chimeras had a lower affinity for the *pYEEI (pY refers to phosphorylated tyrosine) peptide than the wild-type Src-SH2 domain, showing the loop swap alters the disassociation constant of the chimeras for this target peptide. Broad variability was also observed through Ki competition assays. The structures of several chimeras were also visualized through computational molecular modeling, suggesting alterations in the structure of the binding pocket. These results provide further evidence for these loops contributing to the selectivity of SH2 domains, and a better understanding of how these domains function.