Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains—the 7-transmembrane domains—in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.
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All data generated or analysed during this study are included in this published article and its Supplementary Information. The X-ray crystal structures and structure factors of the apo mGlu5 ECD, of the apo mGlu5 ECD in complex with Nb43 and of the mGlu5 ECD in complex with Nb43 and l-quisqualate have been deposited in the Protein Data Bank under accession codes 6N4X, 6N4Y and 6N50, respectively. Cryo-EM maps of apo mGlu5 in nanodiscs, apo mGlu5 in complex with Nb43 and active mGlu5 in complex with Nb43 and l-quisqualate in GDN have been deposited in the Electron Microscopy Data Bank under accession codes EMD-0346, EMD-0347 and EMD-0345, respectively. The atomic coordinates of apo mGlu5 and active mGlu5 in complex with Nb43 and l-quisqualate have been deposited in the Protein Data Bank under accession codes 6N52 and 6N51, respectively.
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The work is supported by National Institutes of Health (NIH) grant R01 NS092695 (G.S., B.K.K. and J.M.M.) and NIH grant R01 NS028471 (B.K.K.). Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) of the NIH under award number T32GM007276 (A.K.). R.F. is funded by grant NNF15OC0015268 from the Novo Nordisk Foundation and the Stanford Bio-X Program. We thank H. Lundbeck for the synthesis of FFMTEB. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the DOE under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIGMS (including P41GM103393). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health. B.K.K. is a Chan Zuckerberg Biohub Investigator.
Nature thanks K. Gregory, J.-P. Pin and J. Rubinstein for their contribution to the peer review of this work.
B.K.K. and T.S.K. are founders of and consultants for ConfometRx, Inc. D.F. and B.S. are employees of ConfometRx, Inc., and M.C. is a former employee of ConfometRx, Inc.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Cartoon view of the apo mGlu5 in complex with Nb43, coloured by b-factors. Notably, the CRDs are diametrically opposed and do not form a stable interface with each other, as reflected by increased b-factors relative to the VFT. b, The Nb43-binding interface, with a 2Fo − Fc map of residues comprising CDR3 at 1σ shown as blue mesh. c, d, Residues involved in Nb43 binding are located on helix L and the L–M loop, and are not conserved. e, The 2Fo − Fc map of the mGlu5 ECD bound to l-quisqualate and Nb43 is shown in grey mesh at 1σ around a cartoon representation of the refined model. The inset shows the quality of density in the l-quisqualate-binding pocket.
a, Two-dimensional negative-stain class averages of apo mGlu5 in LMNG shows several classes with split detergent micelles. b, c, Representative cryo-EM images of apo mGlu5 in nanodiscs (b) and active mGlu5 bound to l-quisqualate and Nb43 in GDN (c). d, e, Cryo-EM class averages of inactive (d) and active mGlu5 bound to l-quisqualate and Nb43 (e). f, Particle angular distribution of the final cryo-EM reconstructions of apo (left) and active (right) mGlu5.
a, b, Processing workflow of apo mGlu5 in MSP1D1 nanodiscs (a) and apo mGlu5 bound to Nb43 in GDN (b).
a, Flow chart indicating the processing of cryo-EM data for mGlu5 in complex with Nb43 and l-quisqualate. b, Three-dimensional density maps of mGlu5 (left) and mGlu5 in complex with Nb43 and l-quisqualate (right), coloured by local resolution. c, ‘Gold standard’ FSC curves from RELION indicate that the maps for both mGlu5 and mGlu5 in complex with Nb43 and l-quisqualate reach nominal resolutions of 4.0 Å at FSC = 0.143.
a–h, Representative cryo-EM densities and fitted atomic models for apo (a, b) and active (c–h) mGlu5. The B and C helices of the VFT and the 7TM domain are highlighted in a for the apo mGlu5 structure, and density and model fits are shown in b. Various helices of the VFT, as well as the ligand-binding pocket, CRD and TM6 are highlighted in c, and their cryo-EM density and fitted models are shown in d–h for active mGlu5 bound to Nb43 and l-quisqualate.
Comparison of intersubunit interfaces in apo and active mGlu5 are shown for apo (top) and active (bottom) mGlu5. Contact regions (purple) show residues within 4 Å of the opposite subunit. Notably, apo mGlu5 lacks any interactions beyond the VFT.
a, The conformation of the VFT and CRD portions of full-length apo mGlu5 obtained using cryo-EM (coloured cartoon) is almost identical to the conformation revealed by the crystal structure of the full-length apo mGlu5 (7TM domain not resolved; grey cartoon). b, Similarly to the apo-state structures, the conformation of the VFT and CRD portions of full-length Nb43-bound, active-state mGlu5 obtained by cryo-EM (coloured cartoon) is almost identical to the conformation revealed by the crystal structure of the ECD bound to l-quisqualate and Nb43. c, d, Despite being bound to a NAM (green) and a PAM (blue), the 7TM domains of in our full-length structures both align almost identically to a crystal structure of a NAM-bound 7TM domain in isolation (c) as well as to each other (d).
Simulation of the apo form of mGlu5 ECD both with and without Nb43. In the absence of Nb43, the ECD relaxes to an open conformation (right) with considerable separation between the bottom lobes of the VFT. The presence of Nb43 restricts opening of the ECD to an intermediate degree (middle) but does not stabilize the compact resting state (left).
a, b, Activation involves a 20 Å translation of the 7TM domains relative to each other (a) followed by a 20° rotation around TM4 (b). The 7TM domains of the apo state are shown as shades of purple, whereas the active state 7TM domains are shown as shades of blue and teal. c, Model of the I7916.56C mutation on the active-state structure shows its ideal positioning for disulfide formation. d, Western blot analysis of disulfide formation involving I7916.56C in both wild type and the C129A background in the presence and absence of the NAM MPEP. Bars in d (bottom) represent mean ± s.e.m. from 8 independent experiments. Statistics were performed using repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test and indicated P values were adjusted to account for multiple comparisons.
This file contains Figures 1-3 and Tables 1-5. Figure 1 – In-vitro characterization of Nb43, Figure 2 - Constitutive activity of ELC2 mutants by IP1 accumulation, Figure 3 – Surface and total expression levels of receptor mutants compared to wild-type. Table 1 - The effect of L-glu as a competitive ligand and the allosteric ligands Nb43 and CDPPB as enhancers of [3H]-quisqualic acid binding, Table 2 - Saturation binding of [3H]-quisqualic acid in presence of vehicle, or a fixed concentration of Nb43 or CDPPB, Table 3 - Potency and Emax estimates of mGlu5 ligands alone and in combination with low receptor expression in presence of EAAT3,Table 4 - Effect of ECL2 deletion on orthosteric and allosteric receptor activation, Table 5 - Estimation of low mGlu5 expression levels by [3H]-MPEP binding
: Activation triggered conformational changes in mGlu5. Map/model fits and morph between apo- and active- mGlu5 upon L-quisqualate binding. Agonist action leads to a compaction of the mGlu5 dimer
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Koehl, A., Hu, H., Feng, D. et al. Structural insights into the activation of metabotropic glutamate receptors. Nature 566, 79–84 (2019). https://doi.org/10.1038/s41586-019-0881-4
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