Substituting alanine for glycine at position 60 in v-HRas 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 GppNpbound 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 RasG60A⅐GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.Ras is an essential component of signal transduction pathways that regulate growth, proliferation, differentiation, and apoptosis in response to the activation of membrane-bound receptors (1, 2). Dominant negative Ras mutants have been widely used to elucidate the role of Ras in a variety of signaling pathways. The asparagine for serine mutant at position 17, RasS17N, is probably the most frequently used dominant negative form of Ras (3) and the success of this mutant popularized the use of dominant negative mutants to study the signaling of other small GTPases. Despite this success, the exact molecular details by which dominant negative GTPases exert their inhibitory function are a matter of debate in the literature. It is widely accepted that RasS17N blocks the ability of endogenous Ras to function by sequestering and depleting the intracellular pool of available guanine nucleotide exchange factor (GEF), 1 thereby blocking the activation of endogenous Ras (4 -6). This argument is supported by the finding that overexpressing a dominant active form of Ras (e.g. RasG12V) or an activator domain usually abolishes the inhibitory effect of dominant negative Ras (3, 7). However, other explanations have been also proposed (8 -9) including low affinity of RasS17N for GTP and the inability of GTP to induce the RasS17N conformation necessary for binding and activating downstream effectors (4, 10). To complicate matters, the S17N mutant of Rap1A, which a priori should behave like RasS17N, is unable to inhibit the activation of Rap1A by its exchange factor, C3G, in vitro (11). Understanding at the molecular level how a dominant negative Ras functions should shed light on its cellular role and help in designing new tools to dissect the signaling of Ras and other small G-proteins. Because it inhibits the activation of endogenous Ras, dissecting the action of a dominant negative Ras should also better our understanding of the reaction of nucleotide exchange. So far, the structure of a dominant negative Ras complex is lacking in the literature.The substitution of alanine for glycine at position 60 in v-H-Ras, v-H-RasG60A, generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 ...