Structure-activity relationships in flexible protein domains: Regulation of rho GTPases by RhoGDI and D4 GDI

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-d iphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions o f both RhoGDI and D4GDI using a series of NMR experiments which include N-1 5 relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro a nd in vivo functional assays have been carried out with N-terminally trunca ted proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abol ish the ability of RhoGDI to inhibit GDP dissociation. The combination of s tructural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-boun d GTPase, but differ in their ability to regulate GTP-bound forms; these fu nctional differences are attributed to the conformational differences of th e N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitor s. Therefore, the two transient helices, appear to be associated with diffe rent biological effects of RhoGDI, providing a clear example of structure-a ctivity relationships in a flexible protein domain. (C) 2001 Academic Press .