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Structure of the K180P mutant of Gi alpha subunit bound to AlF4 and GDP

Overview

Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase, activity that leads to functional deactivation with a rate constant of, approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes, conformational changes in three regions of G alpha, including Switch I and, Switch II. Mutation of G202-->A in Switch II of G alpha(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 G alpha(i1).GTP species. Isothermal, titration calorimetric analysis of the binding of (G202A)G alpha(i1) and, (K180P)G alpha(i1) to the GTPase-activating protein RGS4 indicates that, the G202A mutation stabilizes the pretransition state-like conformation of, G alpha(i1) that is mimicked by the complex of G alpha(i1) with GDP and, magnesium fluoroaluminate, whereas the K180P mutation destabilizes this, state. The crystal structures of (K180P)G alpha(i1) bound to a slowly, hydrolyzable GTP analog, and the GDP.magnesium fluoroaluminate complex, provide evidence that the Mg(2+) 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 alpha 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, alpha(i1), the two events are decoupled kinetically, whereas in the native, protein they are concerted.

About this Structure

1SVK is a Single protein structure of sequence from Rattus norvegicus with MG, ALF and GDP as ligands. Active as Heterotrimeric G-protein GTPase, with EC number 3.6.5.1 Full crystallographic information is available from OCA.

Reference

Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit., Thomas CJ, Du X, Li P, Wang Y, Ross EM, Sprang SR, Proc Natl Acad Sci U S A. 2004 May 18;101(20):7560-5. Epub 2004 May 5. PMID:15128951

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