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

Heterotrimeric G protein alpha (Galpha) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of approximate to2 min(-1) at 30degreesC. GTP hydrolysis causes conformational changes in three regions of Ga, including Switch I and Switch It. Mutation of G202-->A in Switch II of Galpha(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 Galpha(i1).GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)Galpha(i1) and (K18OP)Galpha(i1) to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of Galpha(i1) that is mimicked by the complex of Galpha(i1) with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)Galpha(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 Galpha in which major conformational transitions in Switch I and Switch 11 are obligate events that precede the bond-breaking step in GTIP hydrolysis. In (K180P)Galpha(i1), the two events are decoupled kinetically, whereas in the native protein they are concerted.