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Full-length Gαq–phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

Abstract

Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

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Figure 1: Crystal structure of Gαq–PLCβ3 reveals the C-terminal coiled-coil domain (distal CTD) in the context of a fully active signaling complex.
Figure 2: Structural comparison and sequence conservation of the PLCβ distal CTD.
Figure 3: Cryo-EM projection classification and 3D reconstruction scheme.
Figure 4: The distal CTD interacts with the N-terminal helix of Gαq and the PLCβ3 catalytic core in solution and in crystals.
Figure 5: Model of PLCβ3 regulation by the distal CTD and Gαq.

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Acknowledgements

We thank D. Southworth (Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA) for help with light-scattering experiments. This work was supported by US National Institutes of Health (NIH) grants HL086865, HL071818 and GM081655 (J.J.G.T.), NIH grant DK090165 and the University of Michigan Biological Sciences Scholars Program (G.S.) and an American Heart Association Post-Doctoral Fellowship (A.M.L.). G.S. is supported as a Pew Scholar of Biomedical Sciences. This work used the Cell and Molecular Biology Core of the Michigan Diabetes Research and Training Center, supported by DK20572. This research was supported in part by the NIH through the University of Michigan Cancer Center Support Grant (P30 CA046592). Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and Michigan's Technology Tri-Corridor (grant 085P100817). Use of GM/CA at the Advanced Photon Source at Argonne National Laboratory has been funded in whole or in part with federal funds from the US National Cancer Institute (Y1-CO-1020) and the US National Institute of General Medical Services (Y1-GM-1104). Use of the Advanced Photon Source at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.

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A.M.L., G.S. and J.J.G.T. designed the overall experimental approach. A.M.L. and C.A.B. cloned, expressed and purified all Gαq and PLCβ3 variants and conducted FCPIA assays. A.M.L. carried out all activity-based assays. A.M.L. and C.A.B. crystallized the Gαq–PLCβ3 complex, and A.M.L. determined the structure. S.D. and G.S. carried out all cryo-EM experiments. A.M.L., S.D., G.S. and J.J.G.T. wrote the manuscript.

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Correspondence to John J G Tesmer.

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Lyon, A., Dutta, S., Boguth, C. et al. Full-length Gαq–phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain. Nat Struct Mol Biol 20, 355–362 (2013). https://doi.org/10.1038/nsmb.2497

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