Structure of the G60A Mutant of Ras
2005; Elsevier BV; Volume: 280; Issue: 27 Linguagem: Inglês
10.1074/jbc.m502240200
ISSN1083-351X
AutoresBradley Ford, Karlheinz Skowronek, Sean Boykevisch, Dafna Bar‐Sagi, Nicolas Nassar,
Tópico(s)Cellular transport and secretion
ResumoSubstituting alanine for glycine at position 60 in v-H-Ras generated adominant negative mutant that completely abolished the ability of v-H-Ras totransform NIH 3T3 cells and to induce germinal vesicle breakdown inXenopus oocytes. The crystal structure of the GppNp-bound form ofRasG60A unexpectedly shows that the switch regions adopt an open conformationreminiscent of the structure of the nucleotide-free form of Ras in complexwith Sos. Critical residues that normally stabilize the guanine nucleotide andthe Mg2+ ion have moved considerably. Sos binds to RasG60A but isunable to catalyze nucleotide exchange. Our data suggest that the dominantnegative effect observed for RasG60A·GTP could result from thesequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchangefactor ternary complex. Substituting alanine for glycine at position 60 in v-H-Ras generated adominant negative mutant that completely abolished the ability of v-H-Ras totransform NIH 3T3 cells and to induce germinal vesicle breakdown inXenopus oocytes. The crystal structure of the GppNp-bound form ofRasG60A unexpectedly shows that the switch regions adopt an open conformationreminiscent of the structure of the nucleotide-free form of Ras in complexwith Sos. Critical residues that normally stabilize the guanine nucleotide andthe Mg2+ ion have moved considerably. Sos binds to RasG60A but isunable to catalyze nucleotide exchange. Our data suggest that the dominantnegative effect observed for RasG60A·GTP could result from thesequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchangefactor ternary complex. Ras is an essential component of signal transduction pathways that regulategrowth, proliferation, differentiation, and apoptosis in response to theactivation of membrane-bound receptors(1Vojtek A.B. Der C.J. J.Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar,2Downward J. Nat. Rev.Cancer. 2003; 3: 11-22Crossref PubMed Scopus (2544) Google Scholar). Dominant negative Rasmutants have been widely used to elucidate the role of Ras in a variety ofsignaling pathways. The asparagine for serine mutant at position 17, RasS17N,is probably the most frequently used dominant negative form of Ras(3Feig L.A. Cooper G.M. Mol.Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar) and the success of thismutant popularized the use of dominant negative mutants to study the signalingof other small GTPases. Despite this success, the exact molecular details bywhich dominant negative GTPases exert their inhibitory function are a matterof debate in the literature. It is widely accepted that RasS17N blocks theability of endogenous Ras to function by sequestering and depleting theintracellular pool of available guanine nucleotide exchange factor(GEF), 1The abbreviations used are: GEF, guanine nucleotide exchange factor;Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquidchromatography; DTT, dithiothreitol; WT, wild-type; mant-,N-methylanthraniloyl; RafRBD, Ras binding domain of Raf kinase. therebyblocking the activation of endogenous Ras(4Farnsworth C.L. Feig L.A. Mol. Cell. Biol. 1991; 10: 4822-4829Crossref Scopus (184) Google Scholar, 5Powers S. O'Neill K. Wigler M. Mol. Cell. Biol. 1989; 9: 390-395Crossref PubMed Scopus (131) Google Scholar, 6Stacey D.W. Feig L.A. Gibbs J.B. Mol. Cell. Biol. 1991; 8: 4053-4064Crossref Scopus (136) Google Scholar).This argument is supported by the finding that overexpressing a dominantactive form of Ras (e.g. RasG12V) or an activator domain usuallyabolishes the inhibitory effect of dominant negative Ras(3Feig L.A. Cooper G.M. Mol.Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar,7Schweighoffer F. Cai H. Chevallier-Multon M.C. Fath I. Cooper G. Tocque B. Mol. Cell.Biol. 1993; 13: 39-43Crossref PubMed Scopus (49) Google Scholar). However, other explanationshave been also proposed(8Jung V. Wei W. Ballester R. Camonis J. Mi S. van Aelst L. Wigler M. Broek D. Mol. Cell.Biol. 1994; 14: 3707-3718Crossref PubMed Scopus (51) Google Scholar, 9Cool R.H. Schmidt G. Lenzen C.U. Prinz H. Vogt D. Wittinghofer A. Mol. Cell. Biol. 1999; 19: 6297-6305Crossref PubMed Scopus (61) Google Scholar)including low affinity of RasS17N for GTP and the inability of GTP to inducethe RasS17N conformation necessary for binding and activating downstreameffectors (4Farnsworth C.L. Feig L.A. Mol. Cell. Biol. 1991; 10: 4822-4829Crossref Scopus (184) Google Scholar,10John J. Rensland H. Schlichting I. Vetter I. Borasio G.D. Goody R. Wittinghofer A. J. Biol.Chem. 1993; 268: 923-929Abstract Full Text PDF PubMed Google Scholar). 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 (11van den Berghe N. Cool R.H. Horn G. Wittinghofer A. Oncogene. 1997; 15: 845-850Crossref PubMed Scopus (87) Google Scholar).Understanding at the molecular level how a dominant negative Ras functionsshould shed light on its cellular role and help in designing new tools todissect the signaling of Ras and other small G-proteins. Because it inhibitsthe activation of endogenous Ras, dissecting the action of a dominant negativeRas should also better our understanding of the reaction of nucleotideexchange. So far, the structure of a dominant negative Ras complex is lackingin 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 abolishedthe ability of v-H-Ras to transform NIH 3T3 cells, to induce germinal vesiclebreakdown in Xenopus oocytes, and to activate the Ser/Thr kinaseRaf-1. Moreover, v-H-RasG60A inhibits the ability of v-H-Ras to induce oocytegerminal vesicle breakdown when co-injected(12Sung Y.J. Carter M. Zhong J.M. Hwang Y.W. Biochemistry. 1995; 34: 3470-3477Crossref PubMed Scopus (44) Google Scholar, 13Sung Y.J. Hwang M.C.C. Hwang Y.W. J. Biol. Chem. 1996; 271: 30537-30543Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Hwang M.C. Sung Y.J. Hwang Y.W. J. Biol. Chem. 1996; 271: 8196-8202Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar).As with RasS17N, proper membrane localization of the G60A mutant is necessaryfor its dominant negative effect. Biochemical characterization showed that theG60A mutation does not alter the apparent affinity of v-H-Ras or c-H-Ras forGDP, GTP, the Saccharomyces cerevisae exchange factor SDC25C, or forGAP and NF-1. However, the mutation moderately reduced the ability of SDC25Cto stimulate v-H-Ras exchange and abolished the ability of GAP or NF-1 toaccelerate the rate of GTP hydrolysis. In addition, the G60A mutation slightlyattenuated the binding of Ras to Raf but severely reduced the binding toRalGDS (14Hwang M.C. Sung Y.J. Hwang Y.W. J. Biol. Chem. 1996; 271: 8196-8202Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). How RasG60Areverts the transforming ability of constitutively active Ras is still unclearbut conceptually, this reversion is likely to occur through sequestration ofGEFs and/or downstream effectors into nonproductive complexes. In the present work, we have undertaken the structural and biochemicalanalysis of RasG60A to understand the origin of its dominant negative effects.We show that this mutant adopts a novel open conformation in the GppNp-boundform that bears similarities to the structure of nucleotide-free Ras bound tothe catalytic domain of Sos(15Boriack-Sjodin P.A. Margarit S.M. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (627) Google Scholar). Our structural andbiochemical results suggest a new mechanism for the unique dominant negativeeffect of RasG60A. Crystallization and Structure Determination—Wild-type Rasand RasG60A mutant (residues 1–166) were expressed as His-taggedproteins (pProEX-HTb vector) in the Escherichia coli BL21(DE3) strain(30Hall B.E. Yang S.S. Boriack-Sjodin P.A. Kuriyan J. Bar-Sagi D. J. Biol. Chem. 2001; 276: 27629-27637Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Proteins were purifiedon a Ni-NTA column (Qiagen) followed by a Q-Sepharose (Sigma) and a gelfiltration column. The GDP-bound nucleotide was exchanged to GppNp(31John J. Sohmen R. Feuerstein J. Linke R. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 6058-6065Crossref PubMed Scopus (347) Google Scholar) and exchange wasconfirmed by use of an HPLC C18 reverse phase column. For diffractionexperiments, crystals were grown at 20 °C by mixing 4 μl of 20 mg/mlRasG60A (in 20 mm HEPES, pH 7.5, 0.15 m NaCl, 10mm MgCl2) and 4 μl of a reservoir solution. For theGDP-bound form of RasG60A, the reservoir consisted of 30% (w/v) PEG4000, 0.2m magnesium sulfate, and 0.1 m Tris-HCl, pH 8.0. For theGppNp-bound form of RasG60A, the reservoir consisted of 30% (w/v) PEG2000mme,0.15 m magnesium sulfate, 0.1 m HEPES, pH 7.0, and 0.3%hydrogen peroxide. The GDP-bound form crystallized in space group R32(a = 93.6 Å, c = 121.6 Å) with one copy in theasymmetric unit. The GppNp-bound form crystallized in space group I222(a = 34.3 Å, b = 81.4 Å, c = 121.3Å) with one copy in the asymmetric unit. In each case, data werecollected on beamline X26C at the National Synchrotron Laboratory Source,Brookhaven, on a 2k × 2k CCD detector (ADSC), processed with DENZO, andscaled with SCALE-PACK (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar).The structures of the RasG60A mutant were solved by molecular replacement(33Navaza J. Acta Crystallogr.A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) with the depositedcoordinates of wild-type Ras as search model (Protein Data Bank codes 5P21 and4Q21 (16Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar,17Milburn M.V. Tong L. de Vos A.M. Brünger A. Yamaizumi Z. Nishimura S. Kim S.H. Science. 1990; 247: 939-945Crossref PubMed Scopus (849) Google Scholar)). The GDP- and GppNp-boundstructures were refined with the program CNS(34Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol.Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) or REFMAC(35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol.Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar) to 1.70- and 1.84-Åresolution to final crystallographic residualsR/Rfree of 22.8/23.9 and 18.9/24.4%,respectively. Stereochemistry was checked with the program PROCHECK(36Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Data collection andrefinement statistics are summarized inTable I. The final electrondensity maps of both RasG60A forms show Cys118 to be oxidized.Table IData collection and refinement statisticsGDP-bound formGppNp-bound formResolution range (Å)29.7-1.731.6-1.84Rsym,aRsym = Σi,hkl|〈I(hkl)〉 -Ii(hkl)|/Σi,hklIi(hkl),% overall (last shell)8.2 (56.8)8.7 (57.3)Completeness (%) ,overall (last shell)99.5 (99.8)97.5 (98.9)Multiplicity, overall (last shell)11.5 (7.6)4.4 (4.2)No. of unique reflections22,65515,188Protein atoms1,3251,333Heterogeneous atoms3034Solvent atoms6898B factor (A2), overall (from Wilson plot)38.3 (27.5)28.0 (27.1)RfreebRfree =Σ(hkl)ϵT||Fobs| -|Fcalc||/Σ(hkl)ϵT|Fobs|,where T is the test set(39) obtained by randomlyselecting 10% of the data. Last resolution shell is 1.81-1.70 Å and1.89-1.84 Å for the GDP- and GppNp-bound forms, respectively(%), overall (last resolution shell)23.9 (30.7)24.4 (43.1)Rcryst,cRcryst = Σ(hkl)||Fobs| -|Fcalc||/Σ(hkl)|Fobs|(%), overall (last resolution shell)22.8 (29.6)18.3 (27.9)Root mean square deviation in bond length (Å)0.0070.014Root mean square deviation in bond angle (°)1.21.6Estimated coordinateerrordEstimated coordinate error calculated for the data in the 5.0 Å tothe highest resolution range from the Luzzati/SIGMAA statistics(Å)0.23/0.220.24/0.24RamachandranplaneeMost favored/additional allowed regions(%)91.4/8.694.1/5.9a Rsym = Σi,hkl|〈I(hkl)〉 -Ii(hkl)|/Σi,hklIi(hkl)b Rfree =Σ(hkl)ϵT||Fobs| -|Fcalc||/Σ(hkl)ϵT|Fobs|,where T is the test set(39Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar) obtained by randomlyselecting 10% of the data. Last resolution shell is 1.81-1.70 Å and1.89-1.84 Å for the GDP- and GppNp-bound forms, respectivelyc Rcryst = Σ(hkl)||Fobs| -|Fcalc||/Σ(hkl)|Fobs|d Estimated coordinate error calculated for the data in the 5.0 Å tothe highest resolution range from the Luzzati/SIGMAA statisticse Most favored/additional allowed regions Open table in a new tab NMR Spectroscopy—0.7 ml of a 1 mm solution of theGppNp-bound form of Ras proteins in (10 mm HEPES, 2 mmMgCl2, 0.15 m NaCl, pH 7.5, 10% D2O) was usedfor the NMR spectra. One-dimensional 31P NMR spectra were collectedat 5 °C in a 5-mm broadband probe on a Bruker Avance 700 spectrometer at aphosphorous resonance frequency of 283.42 MHz; 16,384 or 47,104 transientswere recorded after excitation with a 45° pulse at a repetition rate of 2s per transient. A 10-Hz exponential apodization function was applied beforeFourier transformation. The data were processed with the spectrometersoftware. Rate of Nucleotide Exchange—1 μm Ras wasincubated in a solution containing 10 mm HEPES, pH 7.5, 0.15m NaCl, 2 mm β-mercaptoethanol, 2 mmEDTA, 5 μm N-methylanthraniloyl (mant)-nucleotide(Molecular Probes) in a quartz cuvette. The reaction was adjusted to 10mm MgCl2 for 5 min and the decrease in fluorescence wasmonitored following the addition of 500 μm unlabeled GTP. Totalreaction volume was 120 μl; all reactions were conducted at 25 °C, andfluorescence was monitored at each step with an excitation wavelength of 355nm and an emission wavelength of 438 nm (5 nm slit widths on a PerkinElmerLS-50B luminescence spectrometer). For the mant-GTP experiment, change in fluorescence was monitored after theaddition of MgCl2, without addition of unlabeled nucleotide. Forthe Sos-catalyzed reactions, 1 μm of the purified catalyticdomain of Sos (residues 564 to 1049) in 10 mm HEPES, pH 7.5, 0.15m NaCl, and 5% glycerol was added immediately after the unlabeledGppNp. Rate of GTP Hydrolysis—100 μl of 1 mm Ras wasdiluted to 1 ml in a buffer consisting of 10 mm HEPES, pH 7.5, 0.15m NaCl, 5 mm EDTA, 2 mmβ-mercaptoethanol, 10 mm GTP for 20 min at room temperature.Samples were placed on ice for 5 min, then brought to 20 mmMgCl2 and immediately desalted into 10 mm HEPES, pH 7.5,0.15 m NaCl, 2 mm MgCl2 on a AmershamBiosciences 26/10 fast desalting column at 4 °C. Samples were concentratedto 1 ml at 4 °C, placed on ice, and divided into 20 aliquots. The GTPasereaction was started by placing the samples at 37 °C. Each 50-μlreaction was stopped at the appropriate time point by the addition of 5 μlof 0.25% perchloric acid for 30 s, followed by a pH adjustment with 5 μl of0.5 m sodium acetate, pH 5.5. Final sample pH was measured at∼5.5. Samples were centrifuged to remove denatured protein, loaded on a4.6 mm × 25-cm Beckman ultrasphere C18 column equilibrated in 50mm KH2PO4, pH 6.5, 5 mmtetrabutylammonium bromide, 1 mm sodium azide, 6.5% acetonitrile,and eluted isocratically at 1 ml/min. Eluant was monitored between 220 and 320nm and GTP/GDP peaks were identified by comparison with nucleotidestandards. Ras Activation Assay—COS1 cells were transiently transfectedwith the indicated pCGN (37Corbalan-Garcia S. Margarit S.M. Galron D. Yang S.S. Bar-Sagi D. Mol. Cell. Biol. 1998; 18: 880-886Crossref PubMed Scopus (83) Google Scholar)Ras constructs and allowed to express for 24 h. Cells were rinsed withphosphate-free Dulbecco's modified Eagle's medium (Invitrogen) and incubatedwith phosphate-free Dulbecco's modified Eagle's medium supplemented with 1mCi/ml 32PO4 (ICN) for 4 h prior to harvest. Cells weresubsequently washed three times with ice-cold phosphate-buffered saline andlysed in 400 μl of ice-cold buffer containing 50 mm HEPES, pH7.4, 0.1 m NaCl, 1%Triton X-100, 5 mm MgCl2,1 mg/ml fatty acid-free bovine serum albumin, 1 mmphenylmethylsulfonyl fluoride, 1% aprotinin, 10 μg/ml leupeptin, 10μg/ml pepstatin, 5 μg/ml trypsin inhibitor, and 10 mmbenzamidine. Clarified cell lysate was incubated with activated charcoal for 5min on ice. Ras proteins were immunoprecipitated for 40 min at 4 °C withanti-hemagglutinin antibody (12CA5) precoupled to protein A-Sepharose beads.Immune complexes were collected by centrifugation and washed six times inbuffer containing 50 mm HEPES, pH 7.4, 0.5 m NaCl, 0.1%Triton X-100, 5 mm MgCl2, and 0.005% SDS. Boundnucleotides were eluted in 2 mm EDTA, pH 8.0, 2 mmdithiothreitol, and 0.2% SDS for 20 min at 68 °C. Nucleotides wereresolved by thin layer chromatography on polyethyleneimine-cellulose plates(Machery-Nagel) in 0.75 m KH2PO4, pH 3.4, andquantified with a PhosphorImager (Amersham Biosciences). Percentage GTP wascalculated with the formula [(GTP/3)/[(GTP/3) + (GDP/2)]] × 100, toaccount for differences in 32P incorporation withinnucleotides. Raf-RBD Pull-down Assay—100 nm GST-RafRBD (Raf-1residues 51–131) was incubated with Ras proteins at the concentrationsshown in Fig. 7 in 1 ml of asolution containing 10 mm HEPES, pH 7.5, 0.15 m NaCl, 2mm β-mercaptoethanol for 20 min at room temperature withgentle agitation. Glutathione-agarose beads (50 μl of a 50% slurry; Sigma)were added to each tube for an additional 20 min. Beads were pelleted andwashed twice with 1 ml of buffer, then loaded on a 12% SDS-PAGE gel. Blotswere probed with pan-Ras antibody (Santa Cruz FL-189, 1:2000) and horseradishperoxidase-labeled anti-rabbit antibody (Santa Cruz SC-2004, 1:400) for Rasand with anti-GST (Santa Cruz SC-138, 1:800) and horseradishperoxidase-labeled anti-mouse antibody (Southern Biotech 1010–05) forRafRBD. Blots were developed with ECL Plus reagent (Amersham Biosciences) andexposed on a Kodak Image Station 440CF. Sos Pull-down Assay—Assays were performed as with theRaf-RBD pull-downs with the following changes. His6-Sos (100nm) was incubated with Ras at the concentrations shown inFig. 7. Samples were incubatedwith 50 μl of 50% Ni-NTA-agarose beads (Qiagen). Blots were probed as abovefor Ras, then re-probed with His probe antibody (Santa Cruz SC-804, 1:400) andhorseradish peroxidase-labeled anti-rabbit antibody (Santa Cruz SC2004,1:1000) for His6-tagged Sos. Comparison of GppNp-bound RasG60A and Wild-type Ras— We havesolved the crystal structure of the RasG60A mutant bound to the slowlyhydrolysable GTP analog, GppNp. The final model is refined to 1.84-Åresolution with low crystallographic indicators and excellent stereochemistry(Table I). A section of theelectron density around the GppNp is shown inFig. 1A. Glycine 60 in Ras is located in the conserved57DXXG60 motif of switch 2 that is part of asharp flexible turn (L4) connecting strand β3 and helix α2(16Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar). Flexibility in thismotif, which is important for Mg2+ ion coordination andγ-phosphate stabilization(16Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar,17Milburn M.V. Tong L. de Vos A.M. Brünger A. Yamaizumi Z. Nishimura S. Kim S.H. Science. 1990; 247: 939-945Crossref PubMed Scopus (849) Google Scholar), is essential for theproper cycling of Ras. Whereas the overall structures of the GppNp-bound formsof wild-type Ras (hereafter WT-Ras) and RasG60A superpose well outside theswitch regions, switch 2 is extensively reorganized and represents a newconformation that has not been previously observed in structural studies ofGDP- or GTP-bound Ras (Fig.1B). Although switch 2 is poorly ordered in WT-Ras, itsconformation is well defined in RasG60A. Residue 60 seems to be the initiatorof the switch 2 restructuring. The (φ,Ψ) dihedral angles of thisresidue change from (-80°, -10°) in the WT-Ras structure to (-58°,153°) in the RasG60A structure. The same rotation for residue 60 has beenobserved in the RasA59G mutant, which mimics the structure of an intermediatefor GTP hydrolysis (18Hall B.E. Bar-Sagi D. Nassar N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12138-12142Crossref PubMed Scopus (116) Google Scholar). Thischange does not disturb only the immediate surroundings of position 60 butpropagates to the next eight residues (Gly60 to Arg68),which also undergo large shifts in their dihedral angles. One immediateconsequence of switch 2 restructuring is the displacement of switch 2 residuesaway from the nucleotide (Fig.1B). For example, the Cα of residuesGln61 and Ala66 are shifted by 4.8 and 7.3 Å,respectively, relative to their positions in WT-Ras. Another consequence is alarge change in solvent accessibility of switch 2 residues(Fig. 1, C andD). For example, Tyr64 is not solventprotected as in the wild-type structure, but is totally solvent exposed. Theobserved conformation of switch 2 is stabilized by a network of hydrogen bondsbetween the guanidinium group of Arg68 and an anion hole formed bythe main chain carbonyls of Ala59, Ala60, andGlu63. In addition, a water molecule mediates the interaction ofArg68 with the main chain amide of Ser65. Consistentwith previous reports (18Hall B.E. Bar-Sagi D. Nassar N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12138-12142Crossref PubMed Scopus (116) Google Scholar),our results demonstrate that Gly60 serves as a pivot for switch 2conformational changes and rationalize the conservation of a glycine at thisposition in all GTPases. An unexpected feature of the GppNp-bound structure of RasG60A is theremodeling of switch 1 to a conformation that completely pulls it away fromthe nucleotide into the solvent (Fig.2A). For example, the Cα of Phe28, theresidue that normally stabilizes the guanine ring by stacking interactions inall nucleotide-bound forms of Ras, has moved 13.1 Å from its position inWT-Ras. Thr35, which coordinates the Mg2+ ion and theγ-phosphate in all GTP-bound structures of Ras, has also moved 3.5Å (Cα) from the position it occupies in WT-Ras. This movementpropels Thr35 into the solvent(Fig. 1D) and preventsit from directly coordinating the Mg2+ ion and theγ-phosphate. The temperature factors of switch 1 residues 30 to 37 areamong the highest in the final model (50 Å2 versusan average of 28 Å2) but the electron density of this regionis clear enough to trace it. The observed conformation of switch 1 isstabilized by hydrophobic interactions between the phenyl group ofPhe28 and apolar residues provided by crystal packing and by thestrong hydrogen bonds Glu37 makes with Ala59 (2.9Å) and Ser65 of switch 2 (2.6 Å), which are not presentin WT-Ras. The strong interaction between Glu37 andSer65 maintains the C terminus of switch 1 (residues 38–40)in a conformation similar to that of WT-Ras (Figs.1B and2E). Thus, the presentconformation of switch 1 shows that when it adopts an open conformation, Rashas evolved a Phe28- and Thr35-independent way ofstabilizing the nucleotide and the Mg2+ ion. The remodeling of switch 1 and 2 affects nucleotide stabilization andMg2+ ion coordination in an unprecedented way. Specifically, thenucleotide is more solvent exposed in RasG60A than in the wild-type structure(Fig. 1C). Surfaceaccessibility calculations(19Hubbard S.J. Thornton J.M. NACCESS computer program. Department of Biochemistryand Molecular Biology, University College London, 1993Google Scholar) show that thesurface-exposed area of the GppNp has more than doubled between the RasG60A(191 Å2) and the wild-type structure (85Å2). The position of the phenyl group of Phe28 isreplaced in the RasG60A·GppNp structure by Lys147 of theconserved 145SAK147 motif(20Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1844) Google Scholar). The long aliphatic sidechain of Lys147, which stabilizes the phenyl group ofPhe28 by stacking interactions in WT-Ras, now occupies the voidleft by the displacement of Phe28. Consequently, the guanine basein RasG60A is stacked between the long side chains of Lys117 andLys147 (Fig. 1, B andC). The interactions of Asn116 andAsp119 with the guanine base are conserved between WT-Ras andRasG60A. The ribose hydroxyls no longer interact with the protein main chaincarbonyls, instead the OH2′ is within hydrogen-bond distance with theside chain amino group of Lys147(Fig. 1B). As aconsequence, the ribose is more solvent exposed in RasG60A. The α- andβ-phosphates have conserved interactions with the protein(16Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar) with the exception thatthey are more solvent accessible (Fig.1C). The γ-phosphate, which in WT-Ras makes directhydrogen bonds to Lys16 of the P-loop, Thr35 of switch1, and Gly60 of switch 2 is more solvent exposed, does not interactwith Thr35, and interacts with the main chain amide ofAla60 through a water molecule(Fig. 2C). Inaddition, there is no equivalent in the RasG60A structure for the watermolecule responsible for the nucleophilic attack on the γ-phosphate(16Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar). The Mg2+ ionmakes conserved interactions in RasG60A with one important exception. Thehydroxyl group of Thr35 is not coordinating the metal ion, as isthe case in all known GTP-bound structures of Ras, but is 6.4 Å from itsposition in WT-Ras. Instead, a water molecule completes the octahedralcoordination of the Mg2+ (Fig.2D). Thus, the G60A mutation, which confers a dominantnegative characteristic for v-H-Ras, prevents Ras from adopting an activeconformation and results in improper Mg2+-coordination. Comparison of RasG60A·GppNp with Nucleotide-freeRas— The structure of the RasG60A mutant is reminiscent ofnucleotide-free Ras (hereafter NF-Ras) in complex with Sos(15Boriack-Sjodin P.A. Margarit S.M. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (627) Google Scholar), which corresponds to astable intermediate on the path for the catalyzed guanine nucleotide exchangereaction. In both structures, switch 1 adopts an "open"conformation that destabilizes the Phe28/guanine base interactionand the coordination of the Mg2+ ion by Thr35, whereasswitch 2 is displaced from the γ-phosphate. In addition,Tyr64 and Tyr71, which are at the heart of the Ras/Sosinterface, adopt positions that are close to their positions in NF-Ras.However, the conformations of the switch regions in NF-Ras and RasG60Astructures are not identical (Fig.2). In particular, Phe28 and Thr35 are moredistant from the nucleotide-binding site in NF-Ras than in the RasG60Astructure (Fig. 2A).The side chain of Ala59, which in NF-Ras sterically hinders thepositioning of the Mg2+ ion to its binding site, has a wild-typeconformation in the RasG60A structure (Fig.2B). Met67, which is buried at the Ras/Sosinterface, is displaced 4.9 Å in the RasG60A structure from its positionin NF-Ras. Very likely, the presence of the catalytic domain of Sos, whichextensively interacts with residues of both switch regions, is responsible forthe observed differences in the switch regions between RasG60A and NF-Ras. Structure of RasG60A·GDP—To find out whetherthe switch regions adopt an open conformation in the inactive form of RasG60A,we solved its crystal structure. The RasG60A·GDP structure superposeswell on the WT-Ras·GDP including the switch regions(17Milburn M.V. Tong L. de Vos A.M. Brünger A. Yamaizumi Z. Nishimura S. Kim S.H. Science. 1990; 247: 939-945Crossref PubMed Scopus (849) Google Scholar)(Table I). The GDP and theMg2+ ion make conserved interactions with RasG60A including thePhe28/guanine-ring stacking interaction. This observation showsthat the open conformation is only characteristic of the triphosphate-boundform of the nucleotide and not the diphosphate. Solution Studies—To further establish that the differencesin the conformations of the switch regions between WT-Ras and RasG60A exist insolution and are not caused by crystal packing or other artifacts, we comparedthe one-dimensional 31P NMR solution spectra of GppNp-bound WT-Rasand RasG60A. The 31P resonances are sensitive to the environment ofthe bound GppNp and thus should provide information on the conformation of theswitch regions. Fig.3A shows that in the case of the wild-type protein, thereare three distinct resonances for the α- and β-phosphates and asingle resonance for the γ-phosphate. The data are consistent with atleast three distinct states of the nucleotide-protein complex that exist inslow exchange. In the case of RasG60A, there is only a single resonance foreach of the α-, β-, and γ-phosphates. The integratedintensities of the α-, β-, and γ-phosphate resonances areequal. The data are consistent with either one static conformation of thebound GppNp or multiple conformations in fast exchange. The comparison betweenthe two NMR spectra suggests that in solution a large conformational changearound the α- and β-phosphates of the GppNp takes place because ofthe glycine to alanine mutation. One plausible explanation for the observeddifferences in the 31P NMR spectra is that in solution, the switchregions of WT-Ras and RasG60A adopt different conformations. To confirm further that the difference in the switch conformations existsin solution, we used the fluorescent nucleotide derivative mant-GTP. The mantmoiety, which is attached to the ribose hydroxyls, is located in proximity ofthe polypeptide Phe28 to Tyr32. We predicted from theGTP- and GDP-bound structures of RasG60A that follo
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