Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLC gamma, Crk, and Grb2

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X(6)PXXPX(6) to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPX Psi P, whereas Yes SH3 selects Psi XXRPLPXLP, Abl SH3 selects PPX Theta XPPP Psi P, Cortactin SH3 selects +PP Psi PXKPXWL, p53bp2 SH3 selects RPX Psi P Psi R+SXP, PLC gamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects Psi P Psi LP Psi K, and Grb2 N-terminal SH3 selects +Theta DXPLPXLP (where Psi, Theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.