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The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Significance Heterotrimeric G proteins are activated by exchange of a bound GDP for GTP at the active site of the alpha subunit (Gα), 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 Gα 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 (Gα), 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 Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα 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 Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 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 Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.