Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family[S]

The role of surface loops in encoding SH2 domain specificity has been systematically investigated by characterizing a group of loop variants obtained from screening phage-displayed SH2 domain libraries. The reported results support a general role for the EF loop (which connects the β-strands E and F) and the BG loop (which connects the α-helix B and β-strand G) in encoding SH2 specificity, add to our understanding of the mechanism of target sequence recognition by an SH2 domain in cells, and have general implications for the evolution of binding specificity of protein interaction modules. [Display omitted] Highlights •Surface loops play an essential role in SH2 domain specificity.•Diverse specificities may be obtained from a single SH2 domain by combinatorial mutations in the EF and BG loops.•The specificity of a loop mutant correlates with the sequence characteristics of the bait peptide used in its isolation. Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of spe