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

Celestine J. Thomas, Xinlin Du, PiLong Li, Ying Wang, Elliott M. Ross, Stephen R. Sprang

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Abstract

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 Gαi1 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 Gαi1·GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)Gαi1 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 Gαi1 with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)Gαi1 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)Gαi1, the two events are decoupled kinstically, whereas in the native protein they are concerted.

Original languageEnglish (US)
Pages (from-to)7560-7565
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number20
DOIs
StatePublished - May 18 2004

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