title: Conformational basis for SH2-Tyr(P)527 binding in Src inactivation.
abstract: 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.
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston 02881, USA.
authors.forename: Marina K
authors.initials: MK
authors.lastname: Ayrapetov
authors.forename: Yue-Hao
authors.initials: YH
authors.lastname: Wang
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lin
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Gu
authors.forename: Keykavous
authors.initials: K
authors.lastname: Parang
authors.forename: Gongqin
authors.initials: G
authors.lastname: Sun
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D018716:Molecular Mimicry; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides; D014443:Tyrosine; D014364:Tryptophan; D019061:src-Family Kinases