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. 1992 Apr 15;89(8):3649–3653. doi: 10.1073/pnas.89.8.3649

X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.

G G Privé 1, M V Milburn 1, L Tong 1, A M de Vos 1, Z Yamaizumi 1, S Nishimura 1, S H Kim 1
PMCID: PMC48926  PMID: 1565661

Abstract

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  4. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  5. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  6. Brünger A. T., Kuriyan J., Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. doi: 10.1126/science.235.4787.458. [DOI] [PubMed] [Google Scholar]
  7. Brünger A. T., Milburn M. V., Tong L., deVos A. M., Jancarik J., Yamaizumi Z., Nishimura S., Ohtsuka E., Kim S. H. Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4849–4853. doi: 10.1073/pnas.87.12.4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Calés C., Hancock J. F., Marshall C. J., 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. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  9. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  10. Dhar R., Ellis R. W., Shih T. Y., Oroszlan S., Shapiro B., Maizel J., Lowy D., Scolnick E. Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus. Science. 1982 Sep 3;217(4563):934–936. doi: 10.1126/science.6287572. [DOI] [PubMed] [Google Scholar]
  11. Feig L. A., Cooper G. M. Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins. Mol Cell Biol. 1988 Jun;8(6):2472–2478. doi: 10.1128/mcb.8.6.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feuerstein J., Goody R. S., Webb M. R. 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] [Google Scholar]
  13. Frech M., John J., Pizon V., Chardin P., Tavitian A., Clark R., McCormick F., Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. doi: 10.1126/science.2164710. [DOI] [PubMed] [Google Scholar]
  14. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higashi T., Sasai H., Suzuki F., Miyoshi J., Ohuchi T., Takai S., Mori T., Kakunaga T. Hamster cell line suitable for transfection assay of transforming genes. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2409–2413. doi: 10.1073/pnas.87.7.2409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jancarik J., de Vos A., Kim S. H., Miura K., Ohtsuka E., Noguchi S., Nishimura S. Crystallization of human c-H-ras oncogene products. J Mol Biol. 1988 Mar 5;200(1):205–207. doi: 10.1016/0022-2836(88)90345-2. [DOI] [PubMed] [Google Scholar]
  17. 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] [Google Scholar]
  18. Krengel U., Schlichting I., Scherer A., Schumann R., Frech M., John J., Kabsch W., Pai E. F., Wittinghofer A. Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell. 1990 Aug 10;62(3):539–548. doi: 10.1016/0092-8674(90)90018-a. [DOI] [PubMed] [Google Scholar]
  19. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  20. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  21. Pai E. F., Kabsch W., Krengel U., Holmes K. C., 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. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
  22. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Seeburg P. H., Colby W. W., Capon D. J., Goeddel D. V., Levinson A. D. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984 Nov 1;312(5989):71–75. doi: 10.1038/312071a0. [DOI] [PubMed] [Google Scholar]
  24. Tong L. A., de Vos A. M., Milburn M. V., Kim S. H. Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP. J Mol Biol. 1991 Feb 5;217(3):503–516. doi: 10.1016/0022-2836(91)90753-s. [DOI] [PubMed] [Google Scholar]
  25. 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. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  26. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  27. Tsuchida N., Ryder T., Ohtsubo E. Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus. Science. 1982 Sep 3;217(4563):937–939. doi: 10.1126/science.6287573. [DOI] [PubMed] [Google Scholar]
  28. Valencia A., Chardin P., Wittinghofer A., Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991 May 14;30(19):4637–4648. doi: 10.1021/bi00233a001. [DOI] [PubMed] [Google Scholar]
  29. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  30. Xu G. F., Lin B., Tanaka K., Dunn D., Wood D., Gesteland R., White R., Weiss R., Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. doi: 10.1016/0092-8674(90)90149-9. [DOI] [PubMed] [Google Scholar]
  31. de Vos A. M., Tong L., Milburn M. V., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. doi: 10.1126/science.2448879. [DOI] [PubMed] [Google Scholar]

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