Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue (original) (raw)

Nature volume 389, pages 758–762 (1997)Cite this article

Abstract

Small G proteins of the Rho family, which includes Rho, Rac and Cdc42Hs, regulate phosphorylation pathways that control a range of biological functions including cytoskeleton formation and cell proliferation1,2,3,4,5,6,7. They operate as molecular switches, cycling between the biologically active GTP-bound form and the inactive GDP-bound state. Their rate of hydrolysis of GTP to GDP by virtue of their intrinsic GTPase activity is slow, but can be accelerated by up to 105-fold through interaction with rhoGAP, a GTPase-activating protein that stimulates Rho-family proteins8,9. As such, rhoGAP plays a crucial role in regulating Rho-mediated signalling pathways. Here we report the crystal structure of RhoA and rhoGAP complexed with the transition-state analogue GDP.AlF4−at 1.65 Å resolution. There is a rotation of 20 degrees between the Rho and rhoGAP proteins in this complex when compared with the ground-state complex Cdc42Hs.GMPPNP/rhoGAP, in which Cdc42Hs is bound to the non-hydrolysable GTP analogue GMPPNP10. Consequently, in the transition state complex but not in the ground state, the rhoGAP domain contributes a residue, Arg 85GAP, directly into the active site of the G protein. We propose that this residue acts to stabilize the transition state of the GTPase reaction. RhoGAP also appears to function by stabilizing several regions of RhoA that are important in signalling the hydrolysis of GTP.

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

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

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

Additional access options:

Similar content being viewed by others

References

  1. Paterson, H. F. et al. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J. Cell Biol. 111, 1001–1007 (1990).
    Google Scholar
  2. Hall, A. The cellular functions of small GTP-binding proteins. Science 249, 635–640 (1990).
    Google Scholar
  3. Ridley, A. J. & Hall, A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399 (1992).
    Google Scholar
  4. Qiu, R.-G., Chen, J., Kirn, D., McCormick, F. & Symns, M. An essential role for Rac in Ras transformation. Nature 374, 457–459 (1995).
    Article ADS CAS Google Scholar
  5. Ridley, A. J. Rho: theme and variations. Curr. Biol. 6, 1256–1264 (1996).
    Google Scholar
  6. Coso, O. A. et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81, 1137–1146 (1995).
    Google Scholar
  7. Minden, A., Lin, A., Claret, F.-X., Abo, A. & Karin, M. Selective activation of the JNK signaling Cascade and c-Jun Transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81, 1147–1157 (1995).
    Google Scholar
  8. Lancaster, C. et al. Characterization of rhoGAP. J. Biol. Chem. 269, 1137–1142 (1994).
    Google Scholar
  9. Lamarche, N. & Hall, A. GAPs for rho-related GTPases. Trends Genet. 10, 436–440 (1994).
    Google Scholar
  10. Rittinger, K. et al. Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP. Nature 388, 693–697 (1997).
    Article ADS CAS Google Scholar
  11. Wittinghofer, A., Pai, E. & Goody, R. S. Structural and mechanistic aspects of the GTPase reaction of H-ras p21. Handbk Exp. Pharmacol. 108, 195–212 (1993).
    Google Scholar
  12. Barrett, T. et al. The structure of the GTPase-activating domain from p50rhoGAP. Nature 385, 458–461 (1997).
    Article ADS CAS Google Scholar
  13. 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).
    Google Scholar
  14. Coleman, D. E. et al. Structures of active conformations of Giα1and the mechanism of GTP hydrolysis. Nature 265, 1405–1412 (1994).
    CAS Google Scholar
  15. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E. & Sigler, P. B. GTPase mechanism of G proteins from the 1.7 Å crystal structure of transducin-α.GDP.AlF−4. Nature 372, 276–279 (1994).
    Article ADS CAS Google Scholar
  16. Fischer, A. J. et al. X-ray structures of the myosin motor domains of Dictyosteleum discoideum complexed with Mg.ADP.BeFxand Mg.ADP.AlF−4. Biochemistry 34, 8960–8972 (1995).
    Google Scholar
  17. Schlichting, I. & Reinstein, J. Structures of active conformations of UMP-kinase from Dictyostelium discoideum suggest phosphoryl transfer is associative. Biochemistry 36, 9290–9296 (1997).
    Google Scholar
  18. Xu, Y., Morera, S., Janin, J. & Cherfils, J. AlF3mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate and MgADP. Proc. Natl Acad. Sci. USA 94, 3579–3583 (1997).
    Google Scholar
  19. Frech, M. et al. Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: An experimental and theoretical study. Biochemistry 33, 3237–3244 (1994).
    Google Scholar
  20. Pai, E. F. et al. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351–2359 (1990).
    Google Scholar
  21. Hirshberg, M., Stockley, R. W., Dodson, G. & Webb, M. R. The crystal structure of human rac1, a member of the rho-family complexed with a GTP analogue. Nature Struct. Biol. 4, 147–152 (1997).
    Google Scholar
  22. Schmidt, G. et al. Gln63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387, 725–729 (1997).
    Article ADS CAS Google Scholar
  23. Flatau, G. et al. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387, 729–733 (1997).
    Article ADS CAS Google Scholar
  24. Maegley, K. A., Admiral, S. J. & Herschlag, D. Ras-catalysed hydrolysis of GTP: A new perspective from model studies. Proc. Natl Acad. Sci. USA 93, 8160–8166 (1996).
    Google Scholar
  25. McCormick, F. Gasp: not just another oncogene. Nature 340, 678–679 (1989).
    Article ADS CAS Google Scholar
  26. Tesmer, J. J. G., Berman, D. M., Gilman, A. G. & Sprang, S. R. Structure of RGS4 bound to AlF−4-activated Giα1: Stabilisation of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997).
    Google Scholar
  27. Bernstein, B. E., Michels, P. A. M. & Hol, W. G. J. Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation. Nature 385, 275–278 (1997).
    Article ADS CAS Google Scholar
  28. CCP4. The CCP4 suite: programs for X-ray crystallography. Acta Crystallogr. D 50, 760–763 (1994).
  29. Carson, M. Ribbons 2.0. J. Appl. Crystallogr. 24, 958–961 (1991).
    Google Scholar
  30. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).
    Google Scholar

Download references

Author information

Author notes

  1. Katrin Rittinger, Philip A. Walker, John F. Eccleston, Stephen J. Smerdon and Steven J. Gamblin: These authors contributed equally to this work.

Authors and Affiliations

  1. National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
    John F. Eccleston, Stephen J. Smerdon & Steven J. Gamblin

Authors

  1. Katrin Rittinger
    You can also search for this author inPubMed Google Scholar
  2. Philip A. Walker
    You can also search for this author inPubMed Google Scholar
  3. John F. Eccleston
    You can also search for this author inPubMed Google Scholar
  4. Stephen J. Smerdon
    You can also search for this author inPubMed Google Scholar
  5. Steven J. Gamblin
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toStephen J. Smerdon.

Rights and permissions

About this article

Cite this article

Rittinger, K., Walker, P., Eccleston, J. 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). https://doi.org/10.1038/39651

Download citation

This article is cited by