Structural and biochemical characterization of the GTPγS-, GDP · P(i)- , and GDP-bound forms of a GTPase-deficient GLY⁴² → Val mutant of G(iα1)

The Gly⁴² → Val mutant of G(iα1) was characterized structurally and biochemically to elucidate two important features of G(iα1)-catalyzed GTP hydrolysis. The crystal structure of the GTPγS-bound G⁴²VG(iα1) protein demonstrates that the steric bulk of Val⁴² pushes the Gln₂₀₄ residue into a catalytically incompetent conformation, providing a rationale for the diminished GTPase activity of this mutant. The same phenomenon may also account for the diminished GTPase activity of the homologous transforming Gly⁴² → Val mutation in p21(ras). Similarly, the steric bulk of the unique Ser⁴² residue in G(zα) may account for the comparatively slower rate of GTP hydrolysis by this G(α) subunit. The G⁴²VG(iα1) subunit was also characterized structurally in its GDP · P(i)- and GDP-bound states, providing a unique opportunity to view three 'snapshots' of GTP hydrolysis. Hydrolysis of GTP to a transient GDP · P(i)-bound intermediate is associated with substantial conformational changes in the switch II segment of the protein. Eventual release of P(i) results in further removal of switch I from the active site and a highly mobile switch II segment. Despite their disparate biochemical properties, the structural similarity of G⁴²VG(iα1) to the G₂₀₃AG(iα1) mutant in the GDP · P(i)-bound form suggests that both mutations stabilize a conformation of the GDP-P(i)-bound protein that occurs only transiently in the wild-type protein. The structures of the GDP- bound forms of the wild-type and mutant proteins are similar.