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

Abstract

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 β 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 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.G protein | guanine nucleotide exchange factor | double electron electron resonance spectroscopy | tertiary structure | protein dynamics

Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα

“…Structural elements β6-α5, αF, the P-loop and the succeeding α1 helix are all involved. It is likely that destabilization of these interactions promotes nucleotide unbinding, domain separation (Van Eps et al, 2015) and nucleotide release.…”

“…Other evidence implicates these regions as possible loci of direct interaction between Gαi1 and Ric-8A. Specifically, EPR studies show that Gαi1 residues R209 and K180 in Switch I and Switch II, respectively, become partially immobilized upon binding to Ric-8A (Van Eps et al, 2015). The possibility that Switch II is a Ric-8A binding site is also suggested by the observation that a chimera of Gαs that harbors the Switch II sequence of Gαi1 is not protected from ubiquitination by Ric-8B, a Gαs-specific ortholog of Ric-8A (Nagai et al, 2010).…”

“…We observed severe attenuation of amide 15 N- 1 H peaks in the TROSY-HSQC spectrum of Gαi1, and a doubling in the number of protons that become accessible to proton-deuterium exchange relative to Gαi1•GDP. Double electron-electron resonance (DEER) experiments with nitroxide spin-labeled Gαi1 showed extensive broadening in the spectrum of inter-probe distances that accompany displacement of the alpha helical and Ras-like domains of the G protein (Van Eps et al, 2015). In stabilizing the nucleotide-free state, Ric-8A induces or accommodates large changes in the global structure and dynamic behavior of Gαi1.…”

Regulation, Signaling, and Physiological Functions of G-Proteins

“…A mechanistic study of the guanine nucleotide-exchange of GDP for GTP by Ric-8A using SDSL and DEER spectroscopy shed light on some of the structural features of this exchange [62]. Ric-8A binding induces structural displacements in Gα i , resulting in the separation of the α-helical and Ras-like domains.…”

Structural basis for nucleotide exchange in heterotrimeric G proteins

“…S15). The results of a recently published DEER study on GDP-bound Gα i1 in the absence of the βγ subunit also suggest a minority population with separated domains (23). …”