Structural consequences of site-directed mutagenesis in flexible protein domains - NMR characterization of the L(55,56)S mutant of RhoGDI

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal d omain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho G TPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the presen t work we study the effect of this double mutation on the conformational an d dynamic properties of RhoGDI, and describe the weak interaction of the mu tant with Rac1 using chemical shift mapping. We show that the helical conte nt of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus inc reasing the entropic penalty for the immobilization of the helix, and contr ibuting to the loss of binding. In contrast to wild-type RhoGDI, no interac tion with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mu tation on the binding constant (decreased by at least three orders of magni tude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.