Structural and Biochemical Characterization of the GTPγS-, GDP·Pi-, and GDP-Bound Forms of a GTPase-Deficient Gly42 → Val Mutant of Giα1

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Abstract
The Gly42 → Val mutant of Giα1 was characterized structurally and biochemically to elucidate two important features of Giα1-catalyzed GTP hydrolysis. The crystal structure of the GTPγS-bound G42VGiα1 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 p21ras. Similarly, the steric bulk of the unique Ser42 residue in Gzα may account for the comparatively slower rate of GTP hydrolysis by this Gα subunit. The G42VGiα1 subunit was also characterized structurally in its GDP·Pi- and GDP-bound states, providing a unique opportunity to view three “snapshots” of GTP hydrolysis. Hydrolysis of GTP to a transient GDP·Pi-bound intermediate is associated with substantial conformational changes in the switch II segment of the protein. Eventual release of Pi 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 G42VGiα1 to the G203AGiα1 mutant in the GDP·Pi-bound form suggests that both mutations stabilize a conformation of the GDP·Pi-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.