The A326S Mutant of Giα1 as an Approximation of the Receptor-bound State

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approximation of the receptor-α subunit complex, we have considered the properties of A326S Giα1, a mutation discovered originally in Gsα (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164–168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the αi1β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Giα1 for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Giα1, βγ prevents irreversible inactivation of the α subunit at 30 °C. However, the mutation accelerates irreversible inactivation of α at 37 °C despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Giα1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Giα1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Gα subunit in the GDP-bound state.