Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1

Background: Members of the Rho family of small GTP-binding proteins, such a s Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational chang e between the GTP-bound (active) and GDP-bound (inactive) forms. In additio n, most members of the Rho and Rac subfamilies cycle between the cytosol an d membrane, The cytosolic guanine nucleotide dissociation inhibitors, RhoGD Is, regulate both the GDP/GTP exchange cycle and the membrane association/d issociation cycle. Results: We have used NMR spectroscopy and site-directed mutagenesis to ide ntify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the fl exible N-terminal domain of RhoGDI, which has a tendency to form an amphipa thic helix in the free protein, makes a major contribution to the binding e nergy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta 4-beta 5 and beta 6-beta 7 loops , with a slight movement of the 3(10) helix accompanying the interaction. T his binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinc t from this site. Conclusions: Isoprenylated Rac-1 appears to interact with three distinct si tes on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 bind s in a pocket in the folded domain of RhoGDI, This is distinct from the maj or site on this domain occupied by Rac-1 itself, which involves two loops a t the opposite end to the isoprenyl-binding site. It is probable that the f lexible C-terminal region of Rac-1 extends from the site at which Rac-1 con tacts the folded domain of RhoGDI to allow the isoprenyl group to bind in t he pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific int eraction with Rac-1 which contributes substantially to the binding affinity .