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Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

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.

SUBMITTER: McClelland LJ

PROVIDER: S-EPMC7044438 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

McClelland Levi J LJ Zhang Kaiming K Mou Tung-Chung TC Johnston Jake J Yates-Hansen Cindee C Li Shanshan S Thomas Celestine J CJ Doukov Tzanko I TI Triest Sarah S Wohlkonig Alexandre A Tall Gregory G GG Steyaert Jan J Chiu Wah W Sprang Stephen R SR

Nature communications 20200226 1

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 ...[more]

PMID: 32103024

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Project description:Resistance to Inhibitors of Cholinesterase A (Ric-8A) is a 60-kDa cytosolic protein that has chaperone and guanine nucleotide exchange (GEF) activity toward heterotrimeric G protein α subunits of the i, q, and 12/13 classes, catalyzing the release of GDP from Gα and subsequent binding of GTP. In the absence of GTP or GTP analogs, and subsequent to GDP release, Gα forms a stable nucleotide-free complex with Ric-8A. In this study, time-resolved fluorescence anisotropy measurements were employed to detect local motions of Gαi1 labeled at selected sites with Alexa 488 (C5) fluorescent dye (Ax) in the GDP, GTPγS (collectively, GXP), and Ric-8A-bound states. Sites selected for Alexa 488 (C5) derivatization were in the α-helical domain (residue 106), the α-helical domain-Ras-like domain hinge (residue 63), Switch I (residue 180), Switch II (residue 209), Switch III (residue 238), the α4 helix (residue 305), and at the junction between the purine-binding subsite in the β6-α5 loop and the C-terminal α helix (residue 330). In the GXP-bound states, the Alexa fluorophore reports local motions with correlation times ranging from 1.0 to 1.8 ns. The dynamics at Ax180 is slower in Gαi1•GDP than in Gαi1•GTPγS. The reverse is true at Ax209. The order parameters, S(2), for Alexa probes at switch residues are high (0.78-0.88) in Gαi1•GDP and lower (0.67-0.75) in Gαi1•GTPγS, although in crystal structures, switch segments are more ordered in the latter. Local motions at Ax63, Ax180, Ax209, and Ax330 are all markedly slower (2.3-2.8 ns) in Gαi1:Ric-8A than in Gαi1•GXP, and only modest (± 0.1) differences in S(2) are observed at most sites in Gαi1:Ric-8A relative to Gαi1•GXP. The slow dynamics suggests long-range correlated transitions within an ensemble of states and, particularly in the hinge and switch segments that make direct contact with Ric-8A. Induction of Gαi1 structural heterogeneity by Ric-8A provides a mechanism for nucleotide release.

Dataset Information

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

Publications

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

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