title: Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9050, USA.
authors.forename: Celestine J
authors.initials: CJ
authors.lastname: Thomas
authors.forename: Xinlin
authors.initials: X
authors.lastname: Du
authors.forename: PiLong
authors.initials: P
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Elliott M
authors.initials: EM
authors.lastname: Ross
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D011487:Protein Conformation
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D006160:Guanosine Triphosphate