Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Pin, J. P., Galvez, T. & Prézeau, L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol. Ther. 98, 325–354 (2003)
Shigemoto, R. et al. Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci. Lett. 163, 53–57 (1993)
Li, G., Jørgensen, M. & Campbell, B. M. Metabotropic glutamate receptor 5-negative allosteric modulators for the treatment of psychiatric and neurological disorders (2009-July 2013). Pharm. Pat. Anal. 2, 767–802 (2013)
Levenga, J. et al. AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome. Neurobiol. Dis. 42, 311–317 (2011)
Stauffer, S. R. Progress toward positive allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5). ACS Chem. Neurosci. 2, 450–470 (2011)
Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013)
Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)
Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012)
Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013)
Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013)
Siu, F. Y. et al. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444–449 (2013)
Bräuner-Osborne, H., Wellendorf, P. & Jensen, A. A. Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors. Curr. Drug Targets 8, 169–184 (2007)
Kunishima, N. et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971–977 (2000)
Tsuchiya, D., Kunishima, N., Kamiya, N., Jingami, H. & Morikawa, K. Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+. Proc. Natl Acad. Sci. USA 99, 2660–2665 (2002)
Hu, J. & Spiegel, A. M. Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators. J. Cell. Mol. Med. 11, 908–922 (2007)
Conn, P. J., Christopoulos, A. & Lindsley, C. W. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nature Rev. Drug Discov. 8, 41–54 (2009)
Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA 105, 877–882 (2008)
Shibata, Y. et al. Thermostabilization of the neurotensin receptor NTS1. J. Mol. Biol. 390, 262–277 (2009)
Lebon, G., Bennett, K., Jazayeri, A. & Tate, C. G. Thermostabilisation of an agonist-bound conformation of the human adenosine A2A receptor. J. Mol. Biol. 409, 298–310 (2011)
Gasparini, F. et al. [(3)H]-M-MPEP, a potent, subtype-selective radioligand for the metabotropic glutamate receptor subtype 5. Bioorg. Med. Chem. Lett. 12, 407–409 (2002)
Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995)
Fredriksson, R., Lagerström, M. C., Lundin, L. G. & Schiöth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003)
Söding, J. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960 (2005)
Goudet, C. et al. Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors. Proc. Natl Acad. Sci. USA 101, 378–383 (2004)
El Moustaine, D. et al. Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G protein coupling. Proc. Natl Acad. Sci. USA 109, 16342–16347 (2012)
Schwartz, T. W., Frimurer, T. M., Holst, B., Rosenkilde, M. M. & Elling, C. E. Molecular mechanism of 7TM receptor activation—a global toggle switch model. Annu. Rev. Pharmacol. Toxicol. 46, 481–519 (2006)
Hofmann, K. P. et al. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009)
Binet, V. et al. Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors. J. Biol. Chem. 282, 12154–12163 (2007)
Chang, W., Chen, T. H., Pratt, S. & Shoback, D. Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C. J. Biol. Chem. 275, 19955–19963 (2000)
Beqollari, D., Betzenhauser, M. J. & Kammermeier, P. J. Altered G-protein coupling in an mGluR6 point mutant associated with congenital stationary night blindness. Mol. Pharmacol. 76, 992–997 (2009)
Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008)
Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011)
Malherbe, P. et al. Mutational analysis and molecular modeling of the binding pocket of the metabotropic glutamate 5 receptor negative modulator 2-methyl-6-(phenylethynyl)-pyridine. Mol. Pharmacol. 64, 823–832 (2003)
Malherbe, P. et al. Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP. J. Neurochem. 98, 601–615 (2006)
Gregory, K. J. et al. Probing the metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulator (PAM) binding pocket: discovery of point mutations that engender a “molecular switch” in PAM pharmacology. Mol. Pharmacol. 83, 991–1006 (2013)
Pagano, A. et al. The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. J. Biol. Chem. 275, 33750–33758 (2000)
O’Brien, J. A. et al. A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol. Pharmacol. 64, 731–740 (2003)
Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. & Pardo, L. Serine and threonine residues bend α-helices in the χ1 = g− conformation. Biophys. J. 79, 2754–2760 (2000)
Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344, 58–64 (2014)
Robertson, N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36–44 (2011)
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Leslie, A. G. W. & Powell, H. R. Processing diffraction data with Mosflm. Evolving Meth. Macromolecular Crystallogr. 245, 41–51 (2007)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)
We thank R. Owen, J. Waterman and D. Axford at I24, Diamond Light Source, Oxford, UK for technical support. We thank C. G. Tate and other colleagues at Heptares Therapeutics Ltd for suggestions and comments, specifically K. Hollenstein and R. Cheng for contributions to the crystallography, and J. Christopher and G. Brown for sourcing allosteric ligands.
The authors declare no competing financial interests.
Extended data figures and tables
a, The thermal stability of mGlu5 constructs measured using [3H] M-MPEP binding following DDM solubilization. Wild-type full-length mGlu5 (closed circles) has a Tm of 20.6 ± 1.6 °C and mGlu5StaR 569–836-T4L (open circles) has a Tm of 27.2 ± 0.3 °C. The inset shows the wild-type full-length mGlu5 data on a different scale. Data represent the mean ± s.d. from 3 independent experiments. b, mGlu5 crystallization construct (StaR(569–836)-T4L) in schematic representation. Thermostabilizing mutations (green) are: E579A, N667Y, I669A, G675M, T742A, S753A. Residues forming the allosteric pocket are pink. Disordered residues in the structure are grey. The disulphide bond between Cys6443.29–Cys733 is denoted by a dashed yellow line.
Experiments performed in membranes from HEK293T cells transiently expressing mGlu5StaR(569–836)-T4L. a, Saturation binding of [3H]-M-MPEP to mGlu5StaR(569–836)-T4L. Non-specific binding was determined by addition of 0.1 mM MPEP. The data shown (mean ± s.e.m.) is representative of three independent experiments performed in duplicate. Data were fitted globally to a one-site saturation isotherm yielding a Kd of 0.86 ± 0.04 nM and Bmax of 54.7 ± 9.5 pmol mg−1. b, Competition binding. Membranes were incubated with varying concentrations (3 nM–10 µM) of the mGlu5 NAMs fenobam, dipraglurant, MPEP and mavoglurant. Inhibition curves were fitted to a four-parameter logistic equation to determine IC50 values, which were converted to Ki values using Kd values determined by saturation binding and the [3H]-M-MPEP concentration of ∼1 nM. The pKi values obtained are shown in Extended Data Table 2. Data shown are the mean ± s.e.m. of three independent experiments.
a, View of the crystal lattice in the bc and ac planes respectively. The single copy of the mGlu5StaR(569–836)-T4L fusion present in the asymmetric unit is shown as Cα-trace (magenta). Symmetry mates shown as Cα-trace with the receptor TMD coloured green and T4L orange. mGlu5 receptor TMDs stack along the c axis in layers mediated by T4L. b, mGlu5 receptor TMDs pack through an interface mediated by antiparallel TM4–TM4 interactions with the T4L domain swung out towards one side of the receptor. c, The extracellular loops of the mGlu5 TMD are not constrained by packing interactions. Receptor and T4L coloured as in a. Two specific interactions are observed between the mGlu5-TMD N-terminal helical extension and T4L moiety from a symmetry mate. d, The intracellular loops of the mGlu5 TMD are not constrained by packing interactions, receptor and T4L coloured as in a. e, f, Result of 10 ns MD simulation on the mGlu5TMD, models shown are separated by 2 ns of simulation. Cα r.m.s.d. (between starting and final model) = 2.0 Å.
T4L layers are held together by extensive contacts between the T4L moieties. Specifically the N-terminal lobes of T4L (residues 1002–1071) are shifted by up to 3.7 (distance between equivalent Ca positions of E1022) in comparison to the input structure to molecular replacement and refinement (residues 1002-1071 from PDB ID: 3EML). The N-terminal lobes of T4L appear to pivot at V1071 to accommodate packing interactions in the mGlu5StaR(569–836)-T4L monoclinic C121 system, forming an intramolecular β-sheet between residues Y1018–K1019 and Y1018′–K1019′ of a symmetry mate, with additional hydrogen bonds between the back bone of G1012–L1013 and G1012′–L1013′. Hydrogen bonds denoted by dashed red lines, distances measured in angstroms.
IP1 response and number of receptors per cells in HEK293 cells transiently transfected to express wild-type (WT) or the indicated mGlu5 mutants. a, Constitutive activity of wild-type and mutant constructs expressed as percentage of background IP1 levels detected in mock transfected control sample. b, Numbers of expressed receptors per cell were determined using [3H]-M-MPEP binding. Error bars indicate standard error of mean and P values are derived from an unpaired two-tailed t-test. Data are representative of three independent experiments.
About this article
Cite this article
Doré, A., Okrasa, K., Patel, J. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 511, 557–562 (2014) doi:10.1038/nature13396
Scientific Reports (2019)
Current Topics in Medicinal Chemistry (2019)
Functional and pharmacological characterization of an S5 domain hERG mutation associated with short QT syndrome
Frontiers in Pharmacology (2019)