PHAGE DISPLAY SELECTION OF LIGAND RESIDUES IMPORTANT FOR SRC-HOMOLOGY-3 DOMAIN BINDING-SPECIFICITY

The Src homology 3 (SH3) domain is a 50-aa modular unit present in man y cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recogniti on of proline-rich motifs on associated proteins. SH3 domains are impo rtant regulatory elements that have been demonstrated to specify disti nct regulatory pathways important for cell growth, migration, differen tiation, and responses to the external milieu, By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequen ce and to bind to SH3 domains in one of two pseudosymmetrical orientat ions, class I and class II. The class I sites have the consensus seque nce ZP(L/P)PP Psi P whereas the class II consensus is PP Psi PPZ (wher e Psi is a hydrophobic residue and Z is a SH3 domain-specific residue) . We previously showed by M13 phage display that the Src, Fyn, Lyn, an d phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP Psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 doma ins in cells, In the current study, class I and class LT core ligand s equences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues . These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue pre ferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct bindin g specificities. These results indicate that residues that flank the c ore binding sequences shared by many SH3 domains are important determi nants of SH3 binding affinity and selectivity.