The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-'. Mutations of residues in loop Li (Gly-12 and Gly-13), in loop L2 , and in loop LA (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.p21 proteins are the products of the N-, K-, and H-ras genes. They are small guanine nucleotide-binding proteins that are believed to function as signal switch molecules: in the active GTP-bound state, they are switched on; in the GDP-bound state, they are switched off. p21 has an intrinsic GTPase activity, the rate of which determines the lifetime of the active state (5, 10).Mutated forms of the ras genes have been identified in ca. 30% of human tumors. These ras oncogenes code for p21 proteins with point mutations in only three residues: Gly-12, Gly-13, and Gln-61. The mutated proteins have a reduced GTPase activity (5, 8, 9). The GTPase-activating protein (GAP) has been identified by its property of stimulating the conversion of normal p21-GTP to p21-GDP in Xenopus oocytes (32, 41). It has been cloned and sequenced and has a molecular mass of 120 kDa. The fact that it does not stimulate the GTPase activity of oncogenic mutants can explain the transforming properties of those mutants (18,41,42,44).The p21-GAP interaction has not been investigated in detail, and the stimulation of the GTPase by GAP was estimated to be 200-fold (41). By mutational analysis, it has been established that loop L2 of p21 is involved in the interaction with GAP (1,13,18,32). From the threedimensional structure, it would appear that loop L4 could also be involved in this interaction (33,36,37,40). It has also been assumed that the C-terminal domain of GAP is sufficient for the GTPase stimulatory activity (44). We have reevaluated the properties of GAP and show that the maximal stimulation of the GTPase activity by GAP is 105-fold and that mutations in loops Ll, L2, and LA affect the p21-GAP interaction in different ways. We also show that the C-terminal domain of GAP is not sufficient for full activating activity and is apparently modulated by the SH2 (src homologous) domains of GAP.

Ras-related GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states. GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras x GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.

The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic 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 the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.