Structure of the G60A mutant of Ras: implications for the dominant negative effect

Substituting alanine for glycine at position 60 in v-H-Ras generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNp-bound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg(2+) ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A.GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.