Crystal and Cryo-EM Structures of the Cytosolic G Protein Alpha Chaperone and Guanine Nucleotide Exchange Factor Ric-8A Bound to Gαi1

Authors

Levi J. McClelland¹, Kaiming Zhang², Tung-Chung Mou³, Jake Johnston³, Cindee Yates-Hansen¹, Shanshan Li², Celestine J. Thomas¹, Tzanko I. Doukov⁴, Sarah Triest ⁵, Alexandre Wohlkonig⁵, Gregory G. Tall⁶, Jan Steyaert⁷, Wah Chiu⁸, Stephen R. Sprang⁹

Organizations

1. Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA
2. Departments of Bioengineering and James H. Clark Center, Stanford University, Stanford, CA, USA
3. Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA Division of Biological Sciences, University of Montana, Missoula, MT, USA
4. Macromolecular Crystallography Group, Stanford Synchrotron Radiation Light Source, SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA
5. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, BelgiumVIB-VUB Center for Structural Biology, VIB, Brussels, Belgium
6. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA
7. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, BelgiumVIB-VUB Center for Structural Biology, VIB, Brussels, Belgium
8. Graduate Program in Biochemistry and Biophysics, University of Montana, Missoula, MT, USA Biosciences Division, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA
9. Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT, USA Graduate Program in Biochemistry and Biophysics, University of Montana, Missoula, MT, USA Division of Biological Sciences,University of Montana, Missoula, MT, USA

Abstract

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα)¹. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for Gα biogenesis and membrane localization^{2, 3}. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)⁴. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation⁵. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to Gα, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gαi1 (isoform 1 of Gα family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of Gα, disrupting all three switch regions that convey Gα nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of Gα. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of Gα that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes Gα-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

CovalX Technology Used

XL Kit

Outcome

In this article the structural basis of Ric-8A, a cytosolic Guanine Nucleotide Exchange Factor (GEF), and chaperone activities are discussed. Ris-8A is essential to life in multicellular eukaryotes because its chaperone activity is required for G protein alpha subunits (Gα)1 biogenesis and membrane localization.

To understand the structure of Ric-8A, multistep procedures were followed which started with preparing crosslinked Ric-8A and Gαi1. To prepare this crosslinked, each 100 ml of Ric-8A: Gαi1 complex included 20 ml of CovalX K100 reagent (2mg/ml) along with other suberate.

It was found that Ric-8A envelops the GTPase domain of Gα which displaces the helical domain of Gα and C terminal helix, and it also disrupts all three switch regions that convey Gα nucleotide-binding and signaling activity. These cooperative interactions dismantle the GDP binding site, promote GDP release, and protect structural elements of Gα that are dynamic in the nucleotide-free state. It was also discovered that Ric-8A GEF and chaperone activities are enhanced by stabilized Gα-binding elements of Ric-8A structures in vivo phosphorylation.

Source