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

Ravi Kant,Brian Bothner,Celestine J Thomas,Stephen R Sprang,Baisen Zeng

doi:10.7554/elife.19238

Ravi Kant, Brian Bothner + Show 3 more

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https://doi.org/10.7554/elife.19238

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Journal: eLife	Publication Date: Dec 23, 2016
Citations: 26	License type: CC BY 4.0

Affiliation: Montana State University, University of Montana

Abstract

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.

Highlights

Resistance to Inhibitors of Cholinesterase 8A (Ric-8A) is a 59.7 KDa protein that catalyzes the release of GDP from i, q and 13-classes of heterotrimeric G protein a subunits (Ga) in vitro (Tall et al, 2003)
HDX measurements reveal a potential Ga binding site in Ric-8A, which we show by mutagenesis, contributes substantially to Ric-8A guanine nucleotide exchange factor (GEF) activity
In the experiments described here, we used non-myristoylated rat Gai1 and rat (1-491) Ric-8A, in which the 29 C-terminal residues of the full-length protein are absent. This C-terminally truncated Ric-8A exhibits roughly 10% higher GEF activity than the native protein (Thomas et al, 2008) and is referred to as Ric-8A

Summary

Introduction

Resistance to Inhibitors of Cholinesterase 8A (Ric-8A) is a 59.7 KDa protein that catalyzes the release of GDP from i, q and 13-classes of heterotrimeric G protein a subunits (Ga) in vitro (Tall et al, 2003). We show that in the complex with Ric-8A, the peptide amide hydrogen atoms of nucleotide-free Gai1 become accessible to exchange, in amino acids that contact the nucleotide, and in the scaffold of secondary structure elements that encompass and support the nucleotide-binding site.

Results

Conclusion