Uncoupling Conformational Change from GTP Hydrolysis in a Heterotrimeric 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-1at 30°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 Gαi1accelerates 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 Gαi1· GTP species. Isothermal titration calorimetric analysis of the binding of ( G202 A) Gαi1and ( K180 P) Gαi1to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of Gαi1that is mimicked by the complex of Gαi1with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of ( K180 P) Gαi1bound 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 ( K180 P) Gαi1, the two events are decoupled kinetically, whereas in the native protein they are concerted.