STRUCTURE-FUNCTION ANALYSIS OF SH3 DOMAINS - SH3 BINDING-SPECIFICITY ALTERED BY SINGLE AMINO-ACID SUBSTITUTIONS

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with pr oline-rich peptide ligands revealed three binding sites involved in th is interaction: two hydrophobic interactions (between aliphatic prolin e dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pr o motif in the ligand). We examined the importance of the arginine bin ding site of SH3 domains by comparing the binding properties of wild-t ype Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mu tated arginine binding site (D99N) and Abl SH3 mutant constructs engin eered to contain an arginine binding site (T98D and T98D/F91Y). We fou nd that the D99N mutation diminished binding to most Src SH3-binding p roteins in whole cell extracts; however, there was only a moderate red uction in binding to a small subset of Src SH3-binding proteins (inclu ding the Src substrate p68). p68 was shown to contain two Arg-containi ng Asp-99-dependent binding sites and one Asp-99-independent binding s ite which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Ab l SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate t hat Asp-99 is important for Src SH3 binding specificity and that Asp-9 9-dependent binding interactions play a dominant role in Src SH3 recog nition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other in dependent of this residue.