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

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

doi:10.1038/s41467-020-14943-4

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

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

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

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.

Original language	English
Article number	1077
Journal	Nature Communications
Volume	11
Issue number	1
DOIs	https://doi.org/10.1038/s41467-020-14943-4
State	Published - Dec 1 2020

Funding

This research was supported by NIH R01GM105993 (S.R.S.); NIH P41GM103832, R01GM079429, and S10OD021600 (W.C.); NSF 1738547 (T.-C.M); NIH R01-GM088242 (G.G.T.); resources of Instruct-ERIC, part of the European Strategy Forum on Research infrastructures (ESFRI), the Research Foundation-Flanders (FWO) for their support to Nanobody discovery, and the Strategic Research Program (SRP) of the Vrije Universiteit Brussel (S.T. and J.S.). The Integrated Structural Biology Core at the University of Montana Center for Biomolecular Structure and Dynamics is supported by NIH COBRE award P20GM103546 (S.R.S.). We thank Drs. Wuxian Shi and Martin Fuchs at the National Synchrotron Light Source II (NSLS II) for assistance with data collection at the FMX (17-ID-2) beamline, supported by NIH grant P41GM111244 and the Department of Energy (DOE), KP1605010. T.I.D. is supported by the SSRL Structural Molecular Biology Program by the DOE and by NIH grant P41GM103393. Small angle X-ray scattering experiments were conducted at the Advanced Photon Source, operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 with support of NIH grant P41 GM103622 and 1S10OD018090-01 for purchase of the Pilatus 3 1M detector. We thank Dr. Baisen Zeng for assistance with GEF assays and SPR quantitation of Nb-Ric-8A binding.

Funders	Funder number
17-ID-2
1738547
P41GM103393
P41GM103832
DE-AC02-06CH11357, KP1605010
Argonne National Laboratory
S10OD021600, P41GM111244, R01GM088242, 1S10OD018090-01, P20GM103546, P41 GM103622, R01GM105993, R01GM079429

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McClelland, L. J., Zhang, K., Mou, T. C., Johnston, J., Yates-Hansen, C., Li, S., Thomas, C. J., Doukov, T. I., Triest, S., Wohlkonig, A., Tall, G. G., Steyaert, J., Chiu, W., & Sprang, S. R. (2020). Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1. Nature Communications, 11(1), Article 1077. https://doi.org/10.1038/s41467-020-14943-4

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title = "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.",

author = "McClelland, \{Levi J.\} and Kaiming Zhang and Mou, \{Tung Chung\} and Jake Johnston and Cindee Yates-Hansen and Shanshan Li and Thomas, \{Celestine J.\} and Doukov, \{Tzanko I.\} and Sarah Triest and Alexandre Wohlkonig and Tall, \{Gregory G.\} and Jan Steyaert and Wah Chiu and Sprang, \{Stephen R.\}",

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doi = "10.1038/s41467-020-14943-4",

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AU - McClelland, Levi J.

AU - Zhang, Kaiming

AU - Mou, Tung Chung

AU - Johnston, Jake

AU - Yates-Hansen, Cindee

AU - Li, Shanshan

AU - Thomas, Celestine J.

AU - Doukov, Tzanko I.

AU - Triest, Sarah

AU - Wohlkonig, Alexandre

AU - Tall, Gregory G.

AU - Steyaert, Jan

AU - Chiu, Wah

AU - Sprang, Stephen R.

PY - 2020/12/1

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N2 - 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.

AB - 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.

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JO - Nature Communications

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

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