THE A326S MUTANT OF G(I-ALPHA-1) AS AN APPROXIMATION OF THE RECEPTOR-BOUND STATE

Agonist-bound heptahelical receptors activate heterotrimeric G protein s by catalyzing exchange of GDP for GTP on their alpha subunits. In se arch of an approximation of the receptor-alpha subunit complex, we hav e considered the properties of A326S G(i alpha 1), a mutation discover ed originally in G(s alpha) (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 accelerat es dissociation of GDP from the alpha(i1)beta(1)gamma(2) heterotrimer by 250-fold. Nevertheless, affinity of mutant G(i alpha 1) for GTP gam ma S is high in the presence of Mg2+, and the mutation has no effect o n the intrinsic GTPase activity of the alpha subunit. The mutation als o uncouples two activities of beta gamma stabilization of the GDP-boun d alpha subunit (which is retained) and retardation of GDP dissociatio n from the heterotrimer (which is lost). For wild-type and mutant G(i alpha 1), beta gamma prevents irreversible inactivation of the alpha s ubunit at 30 degrees C. However, the mutation accelerates irreversible inactivation of alpha at 37 degrees C despite the presence of beta ga mma, Structurally, the mutation weakens affinity for GTP gamma S by st eric crowding: a 2-fold increase in the number of close contacts betwe en the protein and the purine ring of the nucleotide, By contrast, we observe no differences in structure at the GDP binding site between wi ld-type heterotrimers and those containing A326S G(i alpha 1). However , the GDP binding site is only partially occupied ill crystals of G pr otein heterotrimers containing A326S G(i alpha 1). In contrast to orig inal speculations about the structural correlates of receptor-catalyze d nucleotide exchange, rapid dissociation of GDP can be observed in th e absence of substantial structural alteration of a G(alpha) subunit i n the GDP-bound state.