Logo of embojLink to Publisher's site
EMBO J. Aug 1990; 9(8): 2351–2359.
PMCID: PMC552258

Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.


The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.8M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adari H, Lowy DR, Willumsen BM, Der CJ, McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. [PubMed]
  • Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. [PubMed]
  • Brünger AT, Kuriyan J, Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. [PubMed]
  • Burley SK, Petsko GA. Weakly polar interactions in proteins. Adv Protein Chem. 1988;39:125–189. [PubMed]
  • Calés C, Hancock JF, Marshall CJ, Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. [PubMed]
  • Der CJ, Finkel T, Cooper GM. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. [PubMed]
  • Der CJ, Pan BT, Cooper GM. rasH mutants deficient in GTP binding. Mol Cell Biol. 1986 Sep;6(9):3291–3294. [PMC free article] [PubMed]
  • Dever TE, Glynias MJ, Merrick WC. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. [PMC free article] [PubMed]
  • de Vos AM, Tong L, Milburn MV, Matias PM, Jancarik J, Noguchi S, Nishimura S, Miura K, Ohtsuka E, Kim SH. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. [PubMed]
  • Dombroski AJ, Brennan CA, Spear P, Platt T. Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor. J Biol Chem. 1988 Dec 15;263(35):18802–18809. [PubMed]
  • Durbin RM, Burns R, Moulai J, Metcalf P, Freymann D, Blum M, Anderson JE, Harrison SC, Wiley DC. Protein, DNA, and virus crystallography with a focused imaging proportional counter. Science. 1986 May 30;232(4754):1127–1132. [PubMed]
  • Eccleston JF, Webb MR, Ash DE, Reed GH. EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling. J Biol Chem. 1981 Nov 10;256(21):10774–10777. [PubMed]
  • Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. [PubMed]
  • Fasano O, Aldrich T, Tamanoi F, Taparowsky E, Furth M, Wigler M. Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4008–4012. [PMC free article] [PubMed]
  • Feuerstein J, Kalbitzer HR, John J, Goody RS, Wittinghofer A. Characterisation of the metal-ion-GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O-Mn superhyperfine coupling and by kinetic methods. Eur J Biochem. 1987 Jan 2;162(1):49–55. [PubMed]
  • Feuerstein J, Goody RS, Webb MR. The mechanism of guanosine nucleotide hydrolysis by p21 c-Ha-ras. The stereochemical course of the GTPase reaction. J Biol Chem. 1989 Apr 15;264(11):6188–6190. [PubMed]
  • Gibbs JB, Schaber MD, Allard WJ, Sigal IS, Scolnick EM. Purification of ras GTPase activating protein from bovine brain. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5026–5030. [PMC free article] [PubMed]
  • Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. [PubMed]
  • Halliday KR. Regional homology in GTP-binding proto-oncogene products and elongation factors. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(6):435–448. [PubMed]
  • Jacquet E, Parmeggiani A. Substitution of Val20 by Gly in elongation factor Tu. Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes. Eur J Biochem. 1989 Nov 6;185(2):341–346. [PubMed]
  • John J, Frech M, Wittinghofer A. Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction. J Biol Chem. 1988 Aug 25;263(24):11792–11799. [PubMed]
  • John J, Schlichting I, Schiltz E, Rösch P, Wittinghofer A. C-terminal truncation of p21H preserves crucial kinetic and structural properties. J Biol Chem. 1989 Aug 5;264(22):13086–13092. [PubMed]
  • Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. [PubMed]
  • Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. [PubMed]
  • Kalbitzer HR, Goody RS, Wittinghofer A. Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex. Eur J Biochem. 1984 Jun 15;141(3):591–597. [PubMed]
  • Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. [PubMed]
  • Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. [PubMed]
  • Knowles JR. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev Biochem. 1980;49:877–919. [PubMed]
  • la Cour TF, Nyborg J, Thirup S, Clark BF. Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985 Sep;4(9):2385–2388. [PMC free article] [PubMed]
  • McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989 Jan 13;56(1):5–8. [PubMed]
  • McCormick F, Clark BF, la Cour TF, Kjeldgaard M, Norskov-Lauritsen L, Nyborg J. A model for the tertiary structure of p21, the product of the ras oncogene. Science. 1985 Oct 4;230(4721):78–82. [PubMed]
  • Milburn MV, Tong L, deVos AM, Brünger A, Yamaizumi Z, Nishimura S, Kim SH. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. [PubMed]
  • Neal SE, Eccleston JF, Webb MR. Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site. Proc Natl Acad Sci U S A. 1990 May;87(9):3562–3565. [PMC free article] [PubMed]
  • Pai EF, Kabsch W, Krengel U, Holmes KC, John J, Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. [PubMed]
  • Pizon V, Chardin P, Lerosey I, Olofsson B, Tavitian A. Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene. 1988 Aug;3(2):201–204. [PubMed]
  • Rao R, Pagan J, Senior AE. Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase. J Biol Chem. 1988 Nov 5;263(31):15957–15963. [PubMed]
  • Scherer A, John J, Linke R, Goody RS, Wittinghofer A, Pai EF, Homes KC. Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues. J Mol Biol. 1989 Mar 5;206(1):257–259. [PubMed]
  • Schlichting I, Rapp G, John J, Wittinghofer A, Pai EF, Goody RS. Biochemical and crystallographic characterization of a complex of c-Ha-ras p21 and caged GTP with flash photolysis. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7687–7690. [PMC free article] [PubMed]
  • Schulz GE. Structural and functional relationships in the adenylate kinase family. Cold Spring Harb Symp Quant Biol. 1987;52:429–439. [PubMed]
  • Shirakihara Y, Evans PR. Crystal structure of the complex of phosphofructokinase from Escherichia coli with its reaction products. J Mol Biol. 1988 Dec 20;204(4):973–994. [PubMed]
  • Sigal IS, Gibbs JB, D'Alonzo JS, Temeles GL, Wolanski BS, Socher SH, Scolnick EM. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. [PMC free article] [PubMed]
  • Sigal IS, Marshall MS, Schaber MD, Vogel US, Scolnick EM, Gibbs JB. Structure/function studies of the ras protein. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):863–869. [PubMed]
  • Stryer L, Bourne HR. G proteins: a family of signal transducers. Annu Rev Cell Biol. 1986;2:391–419. [PubMed]
  • Tong L, Milburn MV, de Vos AM, Kim SH. Structure of ras proteins. Science. 1989 Jul 21;245(4915):244–244. [PubMed]
  • Trahey M, McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. [PubMed]
  • Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A. Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 1986 Jun;5(6):1351–1358. [PMC free article] [PubMed]
  • Vogel US, Dixon RA, Schaber MD, Diehl RE, Marshall MS, Scolnick EM, Sigal IS, Gibbs JB. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. [PubMed]
  • Watson HC, Walker NP, Shaw PJ, Bryant TN, Wendell PL, Fothergill LA, Perkins RE, Conroy SC, Dobson MJ, Tuite MF, et al. Sequence and structure of yeast phosphoglycerate kinase. EMBO J. 1982;1(12):1635–1640. [PMC free article] [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...