title: Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.
abstract: The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.
authors.affiliation: Department of Biochemistry, University of Oxford, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Hensmann
authors.forename: G W
authors.initials: GW
authors.lastname: Booker
authors.forename: G
authors.initials: G
authors.lastname: Panayotou
authors.forename: J
authors.initials: J
authors.lastname: Boyd
authors.forename: J
authors.initials: J
authors.lastname: Linacre
authors.forename: M
authors.initials: M
authors.lastname: Waterfield
authors.forename: I D
authors.initials: ID
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)