Invasion of the nucleotide snatchers: Structural insights into the mechanism of G protein GEFs

Although the mechanics differ, GEFs appear to deform their substrates in similar ways. In all cases, switch I is pulled away from switch II and the P loop, exposing the active site; dislocation of switch I also displaces a Mg2+-binding residue, but this in itself should have little affect on GDP affinity. GEFs also restructure and displace the amino terminus of switch II and consequently dismantle the χ-phosphate-binding site, at the same time moving a conserved aspartate residue that serves as a water-mediated Mg2+ ligand. Residues, either from the GEF or the G protein, are positioned to occupy and block the phosphate- and Mg2+-binding sites. To varying extents, the P loop is altered and a conserved lysine residue is extracted from the phosphate-binding site. While structural changes in the P loop may occur as a response to the evacuation of the nucleotide-binding site, it appears, especially in the case of EF-Ts, that it is GEF mediated. These rearrangements, however, leave the purine-binding site relatively undisturbed and easily accessed. Hence, incoming nucleotide can gain some purchase onto the GTP-binding site and complete the exchange reaction. Although the dissociation constants for G·GTP, G·GDP, and G·GEF are subnanomolar, that of the ternary complex with either nucleotide (GEF·G protein·GXP) is micromolar (Klebe et al., 1995). The ternary complex is therefore a viable, yet transient intermediate in the pathway of nucleotide exchange in vivo. The delicate energetic balance between binary and ternary complexes is perhaps controlled by the relative concentrations of the two nucleotides. Clearly, structural changes, which have yet to be characterized, must attend the formation of the ternary complex. It is possible that other GEFs, in particular heterotrimeric G protein receptors, employ exchange mechanisms that differ substantially from those discussed here. Nevertheless, there is sufficient diversity among these to offer new insights into both common and unique properties of GEFs, each of which appears to have arisen independently in evolution, and each of which must catalyze nucleotide exchange in a different functional context.