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

Heterotrimeric G protein (G ) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of 2 min 1 at 30°C. GTP hydrolysis causes conformational changes in three regions of G , including Switch I and Switch II. Mutation of G2023A in Switch II of G 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 K1803P in Switch I increases the rate of conformational change but decreases the GTPase rate, which causes transient but substantial accumulation of a low-fluorescence G i1 GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)G i1 and (K180P)G i1 to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of G i1 that is mimicked by the complex of G i1 with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)G 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 G 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)G i1, the two events are decoupled kinetically, whereas in the native protein they are concerted.