A MODULATOR OF RHO-FAMILY G-PROTEINS, RHOGDI, BINDS THESE G-PROTEINS VIA AN IMMUNOGLOBULIN-LIKE DOMAIN AND A FLEXIBLE N-TERMINAL ARM

Background: The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transf ormation by ras and the organization of the actin cytoskeleton. Additi onally, rac has a role in the regulation of phagocyte NADPH oxidase. G uanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nuc leotide dissociation and by controlling their interaction with membran es. Results: We report the structure of rhoGDI, determined by a combin ation of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of r hoGDI are flexible and unstructured in solution. The 2.5 Angstrom crys tal structure of the folded core of rhoGDI, comprising residues 59-204 , shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 3(10) helix. There is an unu sual pocket between the beta sheets of the immunoglobulin fold which m ay bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it rema ins largely flexible even in the complex. Conclusions: The rhoGDI stru cture is notable for the existence of both a structured and a highly f lexible domain, both of which appear to be required for the interactio n with rac. The immunoglobulin-like fold of the structured domain is u nusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest t hat proteolytic cleavage between the flexible and structured regions o f rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structur e of rhoGDI and the recently reported structure of rabGDI, which perfo rms the same function as rhoGDI for the rab family of small G proteins .