期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：50
页码：E8041-E8050
DOI：10.1073/pnas.1612394113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe -subunit of heterotrimeric G proteins is a molecular switch that mediates a great number of physiological processes such as vision, smelling, and blood pressure regulation. A GTPase-activating protein (GAP) [e.g. regulator of G protein signaling 4 (RGS4) in the case of Gi1] regulates the off-switch by catalyzing GTP hydrolysis. Here, we present the molecular reactions of GAP catalysis at atomic resolution using a combination of FTIR spectroscopy and biomolecular simulations. In contrast to X-ray structures, not GTP analogs but GTP itself is used. This approach is crucial to reveal now a previously undescribed GAP mechanism for G. A key player of the hydrolysis reaction, called the arginine finger, is pushed from a monodentate {gamma}-GTP coordination toward a bidentate -{gamma}-GTP coordination by RGS4, and thereby catalyzes GTP-hydrolysis. 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 G subunit that carries GTP in its active state and GDP in its inactive state. The ability of the G 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 Gi1, which is already an intrinsic part of the catalytic center in G 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 Gi1 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 {gamma}-GTP for the intrinsic Gi1 mechanism and pushed toward a bidentate -{gamma}-GTP coordination for the Gi1{middle dot}RGS4 mechanism. This movement induces a charge shift toward {beta}-GTP, increases the planarity of {gamma}-GTP, and thereby catalyzes the hydrolysis.
