Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gα i1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gα i1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gα i1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.GTPase | FTIR spectroscopy | QM/MM calculations | arginine finger | reaction mechanism
Background: Multiple turnover GTPase assays of G␣ are dominated by nucleotide exchange. Results: FTIR elucidates single turnover rates and individual phosphate vibrations. Conclusion: G␣ i1 -R178S is slowed down in single turnover hydrolysis by 2 orders of magnitude, G␣ i1 -Asp 229 and -Asp 231 are key players in Ras-like/all-␣ domain coordination. Significance: With FTIR on G␣ established, detailed information on the reaction mechanism can be obtained.
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