Structure of the G60A Mutant of Ras

Abstract

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 ...

Pocket-Based Drug Design: Exploring Pocket Space

“…Then, the various conformations are screened out by various binding partners among the conformational ensembles to fulfill the biological functions, whose binding sites undergo the corresponding changes to fit these partners. For the activated Ras protein with GTP binding, the NMR structures (95) without the corresponding protein partner did not present the low-frequency transient pocket (the potential intermediate bound state (87)); this experimental information further confirms this theory. So in order to sample the low-frequency neighboring states similar to the intermediate, maintaining this open conformation of the loop will largely reproduce the bound state.…”

The IQGAP1-Rac1 and IQGAP1-Cdc42 Interactions

“…The results from mutations in switch II were thus unexpected ( Table 2). We mutated all possible residues of switch II to Ala, except Gly-60, which is necessary for the conformational change on GTP binding (49), and Gln-61, which is involved in the GTPase mechanism and is already mutated to Leu in all proteins used in these experiments to stabilize the GTP-bound form. Residues 62-70 in Cdc42 were mutated to alanine, but none significantly affected IQGAP1 binding (Table 2).…”

Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

“…We thought this mutant might represent the intrinsic property of the state 1 structure of RasWT because it has a conservative substitution in Thr-35, the residue whose interaction with the ␥-phosphate constitutes a key structural basis for the state transition. Moreover, the GppNHp-bound form of this mutant had been shown by 31 P NMR to predominantly adopt state 1 (10). A past attempt to solve the crystal structure of H-RasT35S-GppNHp had failed to identify the electron density of both the switch I and switch II regions (10).…”

“…Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the 31 P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions.…”

“…X-ray crystallographic and NMR analyses of H-Ras and Rap1A, alone or in complex with their effectors, revealed that the exchange of GTP for GDP results in allosteric conformational changes in two adjacent regions, termed switch I (residues 32-38) and switch II (residues 60 -75), and enables Ras to execute downstream signaling through direct interaction with its effectors, such as Raf kinases and phosphoinositide 3-kinases (2,3). Recent 31 P NMR spectroscopic studies on Ras unveiled its novel structural feature, the conformational dynamics in the GTP-bound form (4). H-Ras and K-Ras in complex with Mg 2ϩ and a non-hydrolyzable GTP analogue, GppNHp, 3 exhibit equilibrium between two distinct conformational states, state 1 and state 2, which are characterized by different chemical shift values for the resonance of the nucleotide phosphorus atoms of the ␣-and ␥-phosphate groups.…”