Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to G\u03b1i1

Levi J Mcclelland,Shanshan Li,Stephen R Sprang,Cindee Yates-Hansen,Wah Chiu,Tung-Chung Mou,Celestine J Thomas,Tzanko I Doukov,Jake Johnston,Gregory G Tall,Sarah Triest,Kaiming Zhang,Alexandre Wohlkonig,Jan Steyaert

doi:10.1038/s41467-020-14943-4

Abstract

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

Highlights

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone
To stabilize and limit the dynamics of the Ric-8A:Gα complex for crystallographic and cryo-electron microscopy (cryo-EM) experiments, we developed a panel of camelid nanobodies (Nb)[23] that recognize either Gαi[1], Ric-8A, or the complex of the two
The great majority of residues at the Gα contact residues are conserved in Ric-8A and Ric-8B (Supplementary Fig 9a)

Summary

Introduction

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Ric-8A is a cytosolic guanine nucleotide exchange factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα)[1,2]. Ric-8A exhibits GEF and chaperone activity towards Gα of the i, q, and 12/13 classes[1], while Ric-8B performs these functions for Gαs and Gαolf—each in a variety of cellular contexts[11,12]. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation[13].

Methods

Results

Conclusion