PHOSPHOPEPTIDE BINDING TO THE N-TERMINAL SH2 DOMAIN OF THE P85-ALPHA SUBUNIT OF PI-3'-KINASE - A HETERONUCLEAR NMR-STUDY

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosp hatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosin e-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated be ta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDYVPMLDMK) into a solution of the SH2-N domain was monitored usi ng heteronuclear 2D and 3D NMR spectroscopy. 2D-{N-15-H-1} heteronucle ar single-quantum correlation (HSQC) experiments were performed at eac h point of the titration to follow changes in both N-15 and H-1 chemic al 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 {N-15-H-1}- HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the inter nal dynamics of the domain, the magnitude of the {N-15-H-1} heteronucl ear 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 mobi lity of either loops or regions of secondary structure. A mode of pept ide binding that involves little conformational change except in the r esidues directly involved in the 2 binding pockets of the p85 alpha SH 2-N domain is suggested by this study.