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

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.

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Figure 1: a, Overall view of the complex between RhoA.GDP.AlF4−/p50rhoGAP viewed along the heterodimer interface.
Figure 2: Stereo ball-and-stick representation of the complete RhoA.GDP.AlF4−/p50rhoGAP interface viewed in an orientation similar to that in Fig. 1a.
Figure 3: Stereo view of a portion of the final 2 F o − F c electron density map around the nucleotide-binding site on RhoA contoured at 2.0σ, with the final refined atomic model for selected residues from RhoA and GAP superimposed.

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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.

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  1. National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    John F. Eccleston, Stephen J. Smerdon & Steven J. Gamblin

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  1. Katrin Rittinger
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  2. Philip A. Walker
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  3. John F. Eccleston
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Correspondence to Stephen J. Smerdon.

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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

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