Uncoupling conformational change from GTP hydrolysis in a heterotrimetric G protein α-subunit

Heterotrimeric G protein α (Gα) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of ≈2 min^-1 at 3O°C. GTP hydrolysis causes conformational changes in three regions of Gα, including Switch I and Switch II. Mutation of G202→A in Switch II of Gα_i1 accelerates the rates of both GTP hydrolysis and conformational change, which is measured by the loss of fluorescence from Trp-211 in Switch II. Mutation of K180→P in Switch I increases the rate of conformational change but decreases the GTPase rate, which causes transient but substantial accumulation of a low-fluorescence Gα_i1·GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)Gα_i1 and (K180P)Gα _i1 to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of Gα _i1 that is mimicked by the complex of Gα_i1 with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)Gα_i1 bound to a slowly hydrolyzable GTP analog, and the GDP-magnesium fluoroaluminate complex provide evidence that the Mg²⁺ binding site is destabilized and that Switch I is torsionally restrained by the K180P mutation. The data are consistent with a catalytic mechanism for Gα in which major conformational transitions in Switch I and Switch II are obligate events that precede the bond-breaking step in GTP hydrolysis. In (K180P)Gα_i1, the two events are decoupled kinstically, whereas in the native protein they are concerted.