3-DIMENSIONAL STRUCTURES AND PROPERTIES OF A TRANSFORMING AND A NONTRANSFORMING GLYCINE-12 MUTANT OF P21(H-RAS)

The three-dimensional structures and biochemical properties of two mut ants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guan osine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to t he most frequent oncogenic and the only nononcogenic mutation of Gly-1 2, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the a tomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to t he interactions of the aspartic acid side chain with Tyr-32, Gln-61, a nd the gamma-phosphate, which result in reduced mobility of these stru ctural elements. The interaction between the carboxylate group of Asp- 12 and the gamma-phosphate is mediated by a shared proton, which we sh ow by P-31 NMR measurements to exist in solution as well. The structur e of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyr rolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the fa ilure of GAP to stimulate GTP hydrolysis.