Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Ras/Rap effector specificity determined by charge reversal

Nature Structural Biology volume 3, pages 723–729 (1996)Cite this article

Abstract

Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specif guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32–40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31—Lys in Rap, Glu in Ras—which creates a favourable complementary interface for the Ras–Raf interaction.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Nassar, N. et al. The 2.2 Å crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with RaplA and a GTP analogue. Nature 375, 554–560 (1995).

    Article  CAS  Google Scholar 

  2. Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1·8 Å resolution. J. Mol. Biol. 194, 531–544 (1987).

    Article  CAS  Google Scholar 

  3. Weber, P.L., Brown, S.C. & Muller, L. Sequential 1H NMR assignments and secondary structure identification of human ubiquitin. Biochemistry 26, 7282–7290 (1987).

    Article  CAS  Google Scholar 

  4. Di Stefano, D.L. & Wand, A.J. Two-dimensional 1H NMR study of human ubiquitin: a main chain directed assignment and structure analysis. Biochemistry 26, 7272–7281 (1987).

    Article  CAS  Google Scholar 

  5. Derrick, J.P. & Wigley, D.B. The third IgG-binding domain from streptococcal protein G. J. Mol. Biol. 243, 906–918 (1994).

    Article  CAS  Google Scholar 

  6. Orengo, C.A., Jones, D.T. & Thornton, J.M. Protein superfamilies and domain superfolds. Nature 372, 631–634 (1994).

    Article  CAS  Google Scholar 

  7. Tsukihara, T. Structure of the [2Fe-2S] Ferredoxin I from the blue-green Alga Aphanothece sacrum at 2. 2 Å resolution. J. Mol. Biol. 216, 399–410 (1990).

    Article  CAS  Google Scholar 

  8. Derrick, J.P. & Wigley, D.B. Crystal structure of a streptococcal protein G domain bound to an Fab fragment. Nature 359, 752–754 (1992).

    Article  CAS  Google Scholar 

  9. Lian, L.Y., Barsukov, I.L., Derrick, J.P. & Roberts, G.C.K. Mapping the interactions between streptococcal protein G and the Fab fragment of IgG in solution. Nature Struct. Biol. 1, 355–357 (1994).

    Article  CAS  Google Scholar 

  10. 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 3, 201–204 (1988).

    CAS  Google Scholar 

  11. Kawata, M. et al. A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. J Biol. Chem. 263, 18965–18971 (1988).

    CAS  PubMed  Google Scholar 

  12. Ohmori, T., Kikuchi, A., Yamamoto, K., Kirn, S. & Takai, Y. Small molecular weight GTP-binding proteins in human platelet membranes. J. Biol. Chem. 264, 1877–1881 (1989).

    CAS  PubMed  Google Scholar 

  13. Zhang, X.-f. et al. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 364, 308–313 (1993).

    Article  CAS  Google Scholar 

  14. Hofer, F., Fields, S., Schneider, D. & Martin, G.S. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. Proc. Natl. Acad. Sci. USA 91, 11089–11093 (1994).

    Article  CAS  Google Scholar 

  15. Kikuchi, A., Demo, S.D., Ye Zhi-Hai Chen, Y.-W. & Williams, L.T. RalGDS family members interact with the effector loop of ras p21. Mol. Cell. Biol. 14, 7483–7491 (1994).

    Article  CAS  Google Scholar 

  16. Spaargaren, M. & Bischoff, J.R. Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap. Proc. Natl. Acad. Sci. USA 91, 12609–12613 (1994).

    Article  CAS  Google Scholar 

  17. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. & Noda, M. A ras-related gene with transformation suppressor activity. Cell 56, 77–84 (1989).

    Article  CAS  Google Scholar 

  18. Zhang, K., Noda, M., Vass, W.C., Papageorge, A.G. & Lowy, D.R. Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1. Science 249, 162–165 (1990).

    Article  CAS  Google Scholar 

  19. Cook, S.J., Rubinfeld, B., Albert, I. & McCormick, F. RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J. 12, 3475–3485 (1993).

    Article  CAS  Google Scholar 

  20. Marshall, M.S. The effector interactions of p21ras. Trends Biochem. Sci. 18, 250–254 (1993).

    Article  CAS  Google Scholar 

  21. Herrmann, C., Horn, G., Spaargaren, M. & Wittinghofer, A. Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1 A and R-Ras with the putative effector molecules Raf-kinase and Ral-guanine nucleotide exchange factor. J. Biol. Chem. 271, 6794–6800 (1996).

    Article  CAS  Google Scholar 

  22. Marshall, M.S. et al. Identification of amino acid residues required for ras p21 target activation. Mol. Cell. Biol. 11, 3997–4004 (1991).

    Article  CAS  Google Scholar 

  23. Zhang, K. et al. Heterogeneous amino acids in ras and raplA specifying sensitivity to GAP proteins. Science 254, 1630–1634 (1991).

    Article  CAS  Google Scholar 

  24. Shirouzu, M. et al. A glutamic acid residue at position 31 of Ras protein is essential to the signal transduction for neurite outgrowth of PC12 cells and the stimulation of GTPase activity by GAPRAS. Oncogene 7, 475–480 (1992).

    CAS  PubMed  Google Scholar 

  25. Pai, E.F. et al. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351–2359 (1990).

    Article  CAS  Google Scholar 

  26. Lee, B. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400 (1971).

    Article  CAS  Google Scholar 

  27. Programs for Protein Crystallography. Acta Crystallogr. D50 760–763 (1994).

  28. Lawrence, M.C. & Colman, P.M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993).

    Article  CAS  Google Scholar 

  29. Block, C., Janknecht, R., Herrmann, C., Nassar, N. & Wittinghofer, A. Quantitative structure-activity analysis correlating Ras/Raf interaction in vitro to Raf activation in vivo. Nature Struct. Biol. 3, 244–251 (1996).

    Article  CAS  Google Scholar 

  30. Hu, C.-D. et al. Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras. J. Biol. Chem. 270, 30274–30277 (1995).

    Article  CAS  Google Scholar 

  31. Drugan, J.K. et al. Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation. J. Biol. Chem. 271, 233–237 (1996).

    Article  CAS  Google Scholar 

  32. Nur-E-Kamal, M.S.A. & Maruta, H. The role of Gin61 and Glu63 of ras GTPases in their activation by NF1 and ras GAP. Mol. Biol. Cell 3, 1437–1442 (1992).

    Article  CAS  Google Scholar 

  33. White, M.A. et al. Multiple Ras functions can contribute to mammalian cell transformation. Cell 80, 1–20 (1995).

    Article  Google Scholar 

  34. Joneson, T., White, M.A., Wigler, M.H. & Bar-Sagi, D. Stimulation of membrane ruffling and MAP kinase activtion by distinct effectors of RAS. Science 271, 810–812 (1996).

    Article  CAS  Google Scholar 

  35. Akasaka, K. et al. Differential structural requirements for interaction of Ras protein with its distinct downstream effectors. J. Biol. Chem. 271, 5353–5360 (1996).

    Article  CAS  Google Scholar 

  36. Amano, M. et al. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N. Science 271, 648–659 (1996).

    Article  CAS  Google Scholar 

  37. Nonaka, H. et al. A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 14, 5931–5938 (1995).

    Article  CAS  Google Scholar 

  38. Diekmann, D., Nobes, C.D., Burbelo, P.O., Abo, A. & Hall, A. Rac GTPase interacts with GAPs and target proteins through multiple effector sites. EMBO. J. 14, 5297–5305 (1995).

    Article  CAS  Google Scholar 

  39. Dorseuil, O., Reibel, L., Bokoch, G.M., Camonis, J. & Gacon, G. The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1. J. Biol. Chem. 271, 83–88 (1996)

    Article  CAS  Google Scholar 

  40. Herrmann, C., Martin, G. & Wittinghofer, A. Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase, J. Biol. Chem. 270, 2901–2905 (1995).

    Article  CAS  Google Scholar 

  41. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  42. Brünger, T.A. XPLOR version 3.1, Manual, Yale Univ.(1993).

  43. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119 (1991).

    Article  CAS  Google Scholar 

  44. Luzzati, P.V. Traitements statistique des erreurs dans la dètermination des structures cristallines. Acta Crystallogr. 5, 802–810 (1952).

    Article  Google Scholar 

  45. Read, R.J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A42, 140–149 (1986).

    Article  CAS  Google Scholar 

  46. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallog. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  47. Nicholls, A., Sharp, K.A. & Honig, B. Proteinfolding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Fund. Genet. 11, 281 (1991).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-lnstitut für molekulare Physiologie, Abteilung Strukturelle Biologie, Postfach 102664, 44026, Dortmund, Germany

    Nicolas Nassar, Gudrun Horn, Christian Herrmann, Christoph Block & Alfred Wittinghofer

  2. Medizinische Hochschule Hannover, Institut fur molekulare Biologie, D-30623, Hannover, Germany

    Ralf Janknecht

Authors
  1. Nicolas Nassar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gudrun Horn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Herrmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christoph Block
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ralf Janknecht
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alfred Wittinghofer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nassar, N., Horn, G., Herrmann, C. et al. Ras/Rap effector specificity determined by charge reversal. Nat Struct Mol Biol 3, 723–729 (1996). https://doi.org/10.1038/nsb0896-723

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0896-723

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing