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

Small G proteins of the Rho family, which includes Rho, pac and Cdc42Hs, regulate phosphorylation pathways that control a range of biological functions including cytoskeleton formation and cell proliferation(1-7). 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 10(5)-fold through interaction with rhoGAP, a GTPase-activating protein that stimulates Rho-family proteins(8,9). 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 Angstrom 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 GMPPNP(10). Consequently, in the transition state complex but not in the ground state, the rhoGAP domain contributes a residue, Arg85(GAP), 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.