How RhoGDI binds Rho

Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch i n cell signaling, alternating between GTP- and GDP-bound states, with its b iologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor). The crystal structures of R hoA-GDP and of the C-terminal immunoglobulin-like domain of RhoGDI (residue s 67-203) are known, but the mechanism by which the two proteins interact i s not known. The functional human RhoA-RhoGDI complex has been expressed in yeast and crystallized (P6(5)22, unit-cell parameters a = b = 139, c = 253 Angstrom, two complexes in the asymmetric unit). Although diffraction from these crystals extends to 3.5 Angstrom and is highly anisotropic, the expe rimentally phased (MAD plus MIR) electron-density map was adequate to revea l the mutual disposition of the two molecules. The result was validated by molecular-replacement calculations when data were corrected for anisotropy. Furthermore, the N-terminus of RhoGDI (the region involved in inhibition o f nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Db1-like nucleotide-exchange factors. The entrance of the hydro phobic pocket of RhoGDI is 25 Angstrom from the last residue in the RhoA mo del, with its C-terminus oriented to accommodate the geranylgeranyl group w ithout conformational change in RhoA.