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 acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)
Mahadevan
D
D
Macromolecular Structure Laboratory, NCI-FCDRC, ABL-Basic Research Program, Frederick, Maryland 21702.
Thanki
N
N
McPhie
P
P
Beeler
J F
JF
Yu
J C
JC
Wlodawer
A
A
Heidaran
M A
MA
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D002232:Carbocyanines / Q000494:pharmacology; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation / Q000187:drug effects*; D011506:Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013056:Spectrophotometry, Ultraviolet; D020703:ras GTPase-Activating Proteins
D002232:Carbocyanines; D020690:GTPase-Activating Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; C085492:aspartyl-tyrosyl-phosphomethionyl-aspartyl-methionyl-lysine; C012040:stains-all; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D015688:Oncogene Protein pp60(v-src); D002118:Calcium