Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

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.

doi:10.1101/2020.01.04.893156

bioRxiv · 10.1101/2020.01.04.893156

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

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.

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Abstract

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (G)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for G biogenesis and membrane localization2, 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 G, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gi1 (isoform 1 of G 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 G, disrupting all three switch regions that convey G nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of G. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of G that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes G-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

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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.. 2020-01-05. Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1. https://doi.org/10.1101/2020.01.04.893156

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