Crystal Structure of the Rac1−RhoGDI Complex Involved in NADPH Oxidase Activation^,

Abstract

A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

In Vivo Dynamics of Rac-Membrane Interactions

“…This difference may be because GTP-Rac binds less well to GDI, or because, in vivo, GTP-Rac interacts with effector proteins that limit its access to GDI. The latter is based on both structural studies showing that effector interaction sites on Rho GTPases overlap with GDI-binding sites (Abdul-Manan et al, 1999;Mott et al, 1999;Hoffman et al, 2000;Scheffzek et al, 2000;Grizot et al, 2001) and direct measurements (Del Pozo et al, 2002). Although dissociation of G12VRac at membrane protrusions is substantially slower, the rate is not negligible (half-time 173 s), consistent with the substantial amount of cytosolic G12VRac complexed with RhoGDI ( Figure 4F and Del Pozo et al, 2002).…”

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation

“…The 19 amino-acid insert sequence present in Rac1b is positioned immediately downstream of switch II, a region known to be involved in effector binding (Bishop and Hall, 2000) as well as interactions with regulatory proteins such as GEFs and GDIs (Ihara et al, 1998;Worthylake et al, 2000;Grizot et al, 2001;Karnoub et al, 2001). Consistent with this possibility, two recent studies found that Rac1b was impaired in RhoGDI and PAK effector, but not GAP, interaction.…”

Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1

“…Experimental evidence has focused on the Rho family-small GTPase Rac1 as a potential intermediate in the hypoxia signal-transduction pathway. Rac1 regulates assembly of the active NAD(P)H oxidase complex and is recognised as a critical determinant of intracellular redox status [37]. Recent data indicate that Rac1 is required for the induction of HIF-1α protein expression and HIF-1α-dependent gene transcription in response to hypoxia, although Rac1-independent signals are also required for HIF-1α activation under non-hypoxic conditions [11].…”