Abstract
Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8–3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABAB receptor.
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Data availability
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession codes: EMD-30301 (CGP54626-bound GABAB receptor), EMD-30300 (baclofen/BHFF-bound GABAB receptor in the presence of Gi1 protein). The atomic coordinates have been deposited in the Protein Data Bank under accession codes: 7C7S (CGP54626-bound GABAB receptor) and 7C7Q (baclofen/BHFF-bound GABAB receptor in the presence of Gi1 protein).
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Acknowledgements
We are grateful to H. Eric Xu for generously providing the plasmids of the heterotrimeric G protein. We thank S. Chang for technical support in cryo-EM data collection at the Center of Cryo-Electron Microscopy, Zhejiang University; Protein expression and purification were performed at the Protein Facilities, Zhejiang University School of Medicine with the support of C. Ma. J.L. was supported by the Ministry of Science and Technology (2018YFA0507003), the National Natural Science Foundation of China (81720108031, 81872945, 31721002 and 31420103909), the Program for Introducing Talents of Discipline to the Universities of the Ministry of Education (B08029), and the Mérieux Research Grants Program of the Institut Mérieux. Y.Z. was supported by the National Natural Science Foundation of China (81922071), the National Key Basic Research Program of China (2019YFA0508800), Zhejiang Province Natural Science Fund for Excellent Young Scholars (LR19H310001) and the Fundamental Research Funds for the Central Universities (2019XZZX001-01-06).
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C.S. designed the constructs, expressed and purified the antagonist-bound GABAB and the agonist/PAM-stimulated GABAB–Gi1 complex for cryo-EM data collection with the help of S.Z.; C.S., R.Z. and C.L. performed pull-down assay; C.L. and L.-N.C. expressed and purified scFv16; C.X. performed BRET assay; D.-D.S. evaluated the sample by negative-stain EM; C.M. prepared the cryo-EM grids; C.M., D.-D.S. collected the cryo-EM data; C.M. performed cryo-EM map calculation, model building and structure refinement; C.M. and C.S. analyzed the structures and prepared the figures with the help of D.-D.S., Q.S. and Z.J.; Y.Z. and J.L. conceived and supervised the project, analyzed the structures, and wrote the manuscript with inputs from all the authors.
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Mao, C., Shen, C., Li, C. et al. Cryo-EM structures of inactive and active GABAB receptor. Cell Res 30, 564–573 (2020). https://doi.org/10.1038/s41422-020-0350-5
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DOI: https://doi.org/10.1038/s41422-020-0350-5
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