The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in G?i1.

ABSTRACT: 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 G?i1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of G?i1 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 G?s upon binding of Gs to the ?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 G?i and provide insight into the mechanism of action of a nonreceptor G? GEF.

SUBMITTER: Van Eps N 

PROVIDER: S-EPMC4321267 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature