The GTPase-activating protein Rap1GAP uses a catalytic asparagine


Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes1,2. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site3. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Gα proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue4. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 Å. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The structure of Rap1GAP.
Figure 2: Mapping the active site.
Figure 3: Mechanistic analysis of Rap1GAP catalysis.
Figure 4: Implications for tuberin.


  1. 1

    Bos, J. L., de Rooij, J. & Reedquist, K. A. Rap1 signalling: adhering to new models. Nature Rev. Mol. Cell. Biol. 2, 369–377 (2001)

  2. 2

    Hattori, M. & Minato, N. Rap1 GTPase: functions, regulation, and malignancy. J. Biochem. (Tokyo) 134, 479–484 (2003)

  3. 3

    Vetter, I. R. & Wittinghofer, A. The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304 (2001)

  4. 4

    Brinkmann, T. et al. Rap-specific GTPase activating protein follows an alternative mechanism. J Biol. Chem. 277, 12525–12531 (2002)

  5. 5

    Rubinfeld, B. et al. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell 65, 1033–1042 (1991)

  6. 6

    Rubinfeld, B. et al. Localisation of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis. Mol. Cell. Biol. 12, 4634–4642 (1992)

  7. 7

    Hattori, M. et al. Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression. Mol Cell. Biol. 15, 552–560 (1995)

  8. 8

    Gao, Q., Srinivasan, S., Boyer, S. N., Wazer, D. E. & Band, V. The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation. Mol. Cell. Biol. 19, 733–744 (1999)

  9. 9

    Manning, B. D. & Cantley, L. C. Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576 (2003)

  10. 10

    Gao, Q. et al. Human papillomavirus type 16 E6-induced degradation of E6TP1 correlates with its ability to immortalise human mammary epithelial cells. J. Virol. 75, 4459–4466 (2001)

  11. 11

    Ishida, D. et al. Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice. Cancer Cell 4, 55–65 (2003)

  12. 12

    Kraemer, A., Brinkmann, T., Plettner, I., Goody, R. & Wittinghofer, A. Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions. J. Mol. Biol. 324, 763–774 (2002)

  13. 13

    Chabre, M. Aluminofluoride and beryllofluoride complexes: a new phosphate analogs in enzymology. Trends Biochem. Sci. 15, 6–10 (1990)

  14. 14

    Scheffzek, K., Ahmadian, M. R. & Wittinghofer, A. GTPase-activating proteins: helping hands to complement an active site. Trends Biochem. Sci. 23, 257–262 (1998)

  15. 15

    Maheshwar, M. M. et al. The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum. Mol. Genet. 6, 1991–1996 (1997)

  16. 16

    Jones, A. C. et al. Comprehensive mutation analysis of TSC1 and TSC2 and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305–1315 (1999)

  17. 17

    Au, K. S. et al. Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients. Am. J. Hum. Genet. 62, 286–294 (1998)

  18. 18

    Klose, A. et al. Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1. Hum. Mol. Genet. 7, 1261–1268 (1998)

  19. 19

    Xu, X., Wang, Y., Barry, D. C., Chanock, S. J. & Bokoch, G. M. Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator. Biochemistry 36, 626–632 (1997)

  20. 20

    Seewald, M. J., Körner, C., Wittinghofer, A. & Vetter, I. R. RanGAP mediates GTP hydrolysis without an arginine finger. Nature 415, 662–666 (2002)

  21. 21

    De Antoni, A., Schmitzova, J., Trepte, H. H., Gallwitz, D. & Albert, S. Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs. J. Biol. Chem. 277, 41023–41031 (2002)

  22. 22

    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)

  23. 23

    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)

  24. 24

    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)

  25. 25

    Graham, D. L., Eccleston, J. F., Chung, C. W. & Lowe, P. N. Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins. Biochemistry 38, 14981–14987 (1999)

  26. 26

    Goldberg, J. Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis. Cell 96, 893–902 (1999)

  27. 27

    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)

  28. 28

    Rittinger, K., Walker, P. A., Eccleston, J. F., Smerdon, S. J. & Gamblin, S. J. Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389, 758–762 (1997)

  29. 29

    Daumke, O., Wittinghofer, A. & Weyand, M. Purification, crystallisation and preliminary structural characterisation of human Rap1GAP. Acta Crystallogr. D 60, 752–754 (2004)

  30. 30

    Lenzen, C., Cool, R. H. & Wittinghofer, A. Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements. Methods Enzymol. 255, 95–109 (1995)

Download references


We thank A. Krämer for the gift of Rap1-Aedans·GTP. We thank I. Schlichting, W. Blankenfeldt, A. Scheidig, A. Rak, E. Wolf and O. Yildiz for data collection and crystallographic advice and the ESRF beam staff of beamline ID14-1 in Grenoble for support. O.D. thanks the Boehringer Ingelheim Fonds and P.P.C. the International Max-Planck Research School for support.

Author information

Correspondence to Alfred Wittinghofer.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Sequence alignment of the Rap1GAP family and Tuberin. (PDF 36 kb)

Supplementary Figure 2

Superimposition of Rap1GAP and Ras. (JPG 48 kb)

Supplementary Figure 3

Ramachandran plot of the Rap1GAP model. (PDF 34 kb)

Supplementary Figure Legends (DOC 38 kb)

Supplementary Table 1

Phasing statistics of the Rap1GAP model. (PDF 7 kb)

Supplementary Table 2

Refinement statistics of the Rap1GAP model. (PDF 16 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading


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.