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：2015
卷号：112
期号：5
页码：1404-1409
DOI：10.1073/pnas.1423878112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceHeterotrimeric G proteins are activated by exchange of a bound GDP for GTP at the active site of the alpha subunit (G), typically mediated by a transmembrane G protein-coupled receptor (GPCR). Resistance to inhibitors of cholinesterase 8A (Ric-8A), a soluble intracellular protein that is essential for embryonic development, can also catalyze nucleotide exchange and activate G proteins. Here, in a mechanistic study of a nonreceptor exchange factor, site-directed spin labeling and dipolar EPR spectroscopy are used to show that Ric-8A triggers the separation of the Ras-like and helical domains, also observed in GPCR-G protein complexes. This separation and induction of structural plasticity around the nucleotide-binding cavity of G promote nucleotide release. Ric-8A and GPCRs, although structurally unrelated, exhibit distinct but related mechanisms of action. Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (G), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for G subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free G as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gi1 pivot apart to occupy multiple resolved states with displacements as large as 25 [IMG]f1.gif" ALT="A" BORDER="0">. The domain displacement appears to be distinct from that observed in Gs upon binding of Gs to the {beta}2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of G, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gi and provide insight into the mechanism of action of a nonreceptor G GEF.
关键词：G protein ; guanine nucleotide exchange factor ; double electron electron resonance spectroscopy ; tertiary structure ; protein dynamics
