Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.
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