Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP

Abstract

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signalling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 10(5) times. We report here the crystal structure of Cdc42Hs, with the non-hydrolysable GTP analogue GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7A resolution. In the complex Cdc42Hs interacts, mainly through its switch I and II regions, with a shallow pocket on rhoGAP which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit G(i alpha1), we propose that it adopts a different conformation during the catalytic cycle which enables it to stabilize the transition state of the GTP-hydrolysis reaction.

The arginine finger of bacteriophage T7 gene 4 helicase: Role in energy coupling

“…1) closely resembles the arrangement of the arginine fingers that are present in the nucleotide-binding sites of G proteins such as Ras (20,21) and the F 1 -ATPase (17). In the case of G proteins, the arginine is provided by a GTPase-activating protein which, when bound, stimulates GTPase activity 10 5 -fold (21).…”

“…The arginine can reside on a distinct activator protein, as in the case of GAP-Ras (20,21), an adjacent subunit of an oligomeric protein such as the F 1 -ATPase (17), or a distinct domain within the protein itself (22). Arginine fingers contribute to NTP hydrolysis through stabilization of the transition state of the reaction and as a trigger for conformational changes after hydrolysis of dTTP (19,23,24).…”

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

“…Consistent with their high sequence similarity, Rho or Cdc42 superpositions onto Rac exhibited small RMSDs, ranging from 0.74 to 0.86 Angstroms, indicating very close structural similarity. The superposition of Cdc42 and Rac reveals that Rac has identical residues in positions corresponding to those identified as participating in binding of Cdc42 to ARHGAP1 [56], identifying Rac's presumed interaction surface with GAP. Inspections of the superimposed crystal structures shows that in every case GAP and p67 phox TPR bind to distinct, nonoverlapping faces of the Rho or Rac protein; one example illustrating the positions of p67 phox and ARHGAP1 when RhoA and Rac are superimposed is shown in Figure 1.…”

Arhgap36-dependent activation of Gli transcription factors

“…These signaling events may occur within the primary cilium, because the subcellular distributions of Arhgap36, Sufu, and Gli converge at this subcellular compartment and Arhgap36-induced Gli activation requires Kif3a Rho GAP domain-containing proteins typically stimulate guanine nucleotide hydrolysis to generate inactive, GDP-bound forms of Rho GTPase (40), and Arhgap36 could similarly inhibit a Rho signaling protein that promotes Sufu activity. However, Arhgap36 lacks the "arginine finger" motif that stimulates GTPase hydrolysis (25), and GAP domain mutants can retain Gli-inducing activity. Arhgap36 may therefore act through a noncatalytic mechanism, as has been observed with the p85 subunit of phosphatidylinositol 3-kinase, the phosphatidylinositol-4,5-bisphosphate 5-phosphatase OCRL, and n-chimaerin (41)(42)(43).…”

“…Although Arhgap36 is predicted to be a Rho GAP family member, it lacks the "arginine finger" motif that participates in Rho GTPase activity (25). The replacement of this structural element with a threonine (T246) suggests that catalytic GAP domain function may not be required for Arhgap36-induced Gli activation.…”