COMPARISON OF CALCIUM-DEPENDENT CONFORMATIONAL-CHANGES IN THE N-TERMINAL SH2-DOMAINS OF P85 AND GAP DEFINES DISTINCT PROPERTIES FOR SH2-DOMAINS

Src-homology region 2 (SH2) domains are stretches of about 100 amino a cids which are found to be structurally conserved in a number of signa ling molecules. These regions have been shown to bind with high affini ty to phosphotyrosine residues within activated receptor tyrosine kina ses. Here we report the bacterial expression and purification of indiv idual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase ( PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) i n amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dy e known to bind to calcium binding proteins. This effect was not obser ved for the GAPNSH2 domain. Circular dichroism analysis of the N-termi nal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the p resence of increasing calcium concentrations. Moreover, the conformati onal change of p85NSH2 induced by calcium could be replicated by addit ion of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-P DGFR binding site for p85. Limited proteolysis studies showed a signif icant calcium-dependent increase in protection of p85NSH2 but not GAPN SH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown t o substitute for calcium, could also protect the p85NSH2 domain from p roteolysis even at 10-fold Iower concentrations. In vitro binding stud ies using purified preparations of activated alpha-PDGFR show that cal cium did not affect the binding of GAPNSH2 domains to activated alpha- PDGFR. In striking contrast, we observed a marked increase in binding of p85NSH2 domains to activated alpha-PDGFR in the presence of calcium ions. Sequence comparisons and molecular modeling of the p85NSH2 doma in based on the v-Src SH2 domain structure show a conserved arrangemen t of oxygen ligands contributing to two potential calcium binding site s within the p85NSH2 domain. These have been rationalized to be surfac e loop regions, i.e., loops 2 and 4 and loops 6, 7, and 8, close to th e N- and C-terminal alpha-helical regions, respectively. Together, our findings suggest that the conformation of p85NSH2, but not that of GA PNSH2, is modulated by the presence of calcium ions. This implies that calcium ions may regulate PI-3K (p85alpha) binding to alpha-PDGFR in vivo and suggests that the NSH2 domains of p85 and GAP may have distin ct functions in receptor-mediated signal transduction.