p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues

Previous structural studies of RasGAP have failed to clearly localize sites of Pas interaction to individual amino acids, Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Pas GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Pas interactions, However, Arg(786), Lys(831), and Arg(925) were observed to be essential for binding td Ras-GTP but not for protein structure. RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Pas. This is the first example of a conserved Pas interaction motif in distinct Pas effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg(786), Lys(831), and Arg(925) residues form salt bridges with the conserved, negatively charged residues in the Pas effector loop.