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.

RanGAP mediates GTP hydrolysis without an arginine finger

Abstract

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called ‘arginine finger’ into the phosphate-binding pocket1,2. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells3. Ran–GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP4. Here we present the three-dimensional structure of a Ran–RanBP1–RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Structure of the ternary complex.
Figure 2: Details of the Ran–RanGAP interface.
Figure 3: The active site.
Figure 4: Position of the catalytic glutamine.

References

  1. 1

    Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Rittinger, K. et al. Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389, 758–762 (1997).

    ADS  CAS  Article  Google Scholar 

  3. 3

    Görlich, D. & Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 (1999).

    Article  Google Scholar 

  4. 4

    Bischoff, F. R., Krebber, H., Smirnova, E., Dong, W. & Ponstingl, H. Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J. 14, 705–715 (1995).

    CAS  Article  Google Scholar 

  5. 5

    Bischoff, F. R. & Görlich, D. RanBP1 is crucial for the release of RanGTP from importin beta-related nuclear transport factors. FEBS Lett. 419, 249–254 (1997).

    CAS  Article  Google Scholar 

  6. 6

    Becker, J. et al. RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae. J. Biol. Chem. 270, 11860–11865 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Hillig, R. C. et al. The crystal structure of rna1p: a new fold for a GTPase-activating protein. Mol. Cell. 3, 781–791 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Vetter, I. R., Arndt, A., Kutay, U., Görlich, D. & Wittinghofer, A. Structural view of the Ran-Importin β interaction at 2.3 Å resolution. Cell 97, 635–646 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Chook, Y. M. & Blobel, G. Structure of the nuclear transport complex karyopherin-β2-Ran.GppNHp. Nature 399, 230–237 (1999).

    ADS  CAS  Article  Google Scholar 

  10. 10

    Haberland, J., Becker, J. & Gerke, V. The acidic C-terminal domain of rna1p is required for the binding of Ran.GTP and for RanGAP activity. J. Biol. Chem. 272, 24717–24726 (1997).

    CAS  Article  Google Scholar 

  11. 11

    Lounsbury, K. M., Richards, S. A., Carey, K. L. & Macara, I. G. Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization. J. Biol. Chem. 271, 32834–32841 (1996).

    CAS  Article  Google Scholar 

  12. 12

    Vetter, I. R., Nowak, C., Nishimoto, T., Kuhlmann, J. & Wittinghofer, A. Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature 398, 39–46 (1999).

    ADS  CAS  Article  Google Scholar 

  13. 13

    Richards, S. A., Lounsbury, K. M. & Macara, I. G. The C terminus of the nuclear RAN/TC4 GTPase stabilizes the GDP-bound state and mediates interactions with RCC1, RAN-GAP, and HTF9A/RANBP1. J. Biol. Chem. 270, 14405–14411 (1995).

    CAS  Article  Google Scholar 

  14. 14

    Scheffzek, K., Klebe, C., Fritz-Wolf, K., Kabsch, W. & Wittinghofer, A. Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form. Nature 374, 378–381 (1995).

    ADS  CAS  Article  Google Scholar 

  15. 15

    Haberland, J. & Gerke, V. Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation. Biochem. J. 343, 653–662 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Nassar, N., Hoffman, G. R., Manor, D., Clardy, J. C. & Cerione, R. A. Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP. Nature Struct. Biol. 5, 1047–1052 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Tesmer, J. J., Berman, D. M., Gilman, A. G. & Sprang, S. R. Structure of RGS4 bound to AlF4--activated Giα1: stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Der, C. J., Finkel, T. & Cooper, G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell 44, 167–176 (1986).

    CAS  Article  Google Scholar 

  19. 19

    Klebe, C., Bischoff, F. R., Ponstingl, H. & Wittinghofer, A. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry 34, 639–647 (1995).

    CAS  Article  Google Scholar 

  20. 20

    Albert, S., Will, E. & Gallwitz, D. Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases. EMBO J. 18, 5216–5225 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Ahmadian, M. R., Stege, P., Scheffzek, K. & Wittinghofer, A. Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nature Struct. Biol. 4, 686–689 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Graham, D. L., Eccleston, J. F. & Lowe, P. N. The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminium fluoride. Biochemistry 38, 985–991 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Berman, D. M., Wilkie, T. M. & Gilman, A. G. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein α subunits. Cell 86, 445–452 (1996).

    CAS  Article  Google Scholar 

  24. 24

    Maegley, K. A., Admiraal, S. J. & Herschlag, D. Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. Proc. Natl Acad. Sci. USA 93, 8160–8166 (1996).

    ADS  CAS  Article  Google Scholar 

  25. 25

    Allin, C., Ahmadian, M. R., Wittinghofer, A. & Gerwert, K. Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time. Proc. Natl Acad. Sci. USA 98, 7754–7759 (2001).

    ADS  CAS  Article  Google Scholar 

  26. 26

    Prakash, B., Renault, L., Praefcke, G. J., Herrmann, C. & Wittinghofer, A. Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism. EMBO J. 19, 4555–4564 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystrallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  29. 29

    Jones, T. A. & Kjeldgaard, M. Electron-density map interpretation. Methods Enzymol. 277, 173–208 (1997).

    CAS  Article  Google Scholar 

  30. 30

    Brunger, A. T. et al. Crystallography and NMR system (CNS): A new software system for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. Alt for atomic absorption spectroscopy; staff of the European Molecular Biology Laboratory/ESRF for access and support at beam lines ID13 and ID14; R. Hillig for the coordinates of uncomplexed RanGAP before release; and the Deutsche Forschungsgemeinschaft (DFG) for a grant to I.R.V.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Alfred Wittinghofer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Seewald, M., Körner, C., Wittinghofer, A. et al. RanGAP mediates GTP hydrolysis without an arginine finger. Nature 415, 662–666 (2002). https://doi.org/10.1038/415662a

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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