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The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in G?i1.

ABSTRACT: Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (G?), 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 G? subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free G? 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 G?i1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of G?i1 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 G?s upon binding of Gs to the ?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 G?, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of G?i and provide insight into the mechanism of action of a nonreceptor G? GEF.

SUBMITTER: Van Eps N

PROVIDER: S-EPMC4321267 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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

Van Eps Ned N Thomas Celestine J CJ Hubbell Wayne L WL Sprang Stephen R SR

Proceedings of the National Academy of Sciences of the United States of America 20150120 5

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 ...[more]

PMID: 25605908

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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

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in G?i1.

Publications

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

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