Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain

Nature volume 511, pages 557–562 (2014)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic and ribbon representation of the mGlu5 structure.
Figure 2: The narrow entrance to the allosteric pocket in mGlu5.
Figure 3: Superposition and comparison of mGlu5 with rhodopsin and CRF1R.
Figure 4: The ‘ionic-lock’ motifs in mGlu5 and rhodopsin.
Figure 5: Molecular details of further conserved sequence motifs in mGlu5 and comparison to rhodopsin.
Figure 6: The mGlu5 allosteric modulator binding site.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Co-ordinates and structure factors have been deposited in the Protein Data Bank under the accession code 4OO9.

References

  1. 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)

    Article  CAS  PubMed  Google Scholar 

  2. Shigemoto, R. et al. Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci. Lett. 163, 53–57 (1993)

    Article  CAS  PubMed  Google Scholar 

  3. 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)

    Article  CAS  PubMed  Google Scholar 

  4. Levenga, J. et al. AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome. Neurobiol. Dis. 42, 311–317 (2011)

    Article  CAS  PubMed  Google Scholar 

  5. Stauffer, S. R. Progress toward positive allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5). ACS Chem. Neurosci. 2, 450–470 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012)

    Article  CAS  PubMed  Google Scholar 

  9. Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Siu, F. Y. et al. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444–449 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  12. 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)

    Article  PubMed  Google Scholar 

  13. Kunishima, N. et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971–977 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. 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)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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)

    Article  CAS  Google Scholar 

  17. 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)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shibata, Y. et al. Thermostabilization of the neurotensin receptor NTS1. J. Mol. Biol. 390, 262–277 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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)

    Article  CAS  PubMed  Google Scholar 

  21. 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)

    Article  CAS  Google Scholar 

  22. 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)

    Article  CAS  PubMed  Google Scholar 

  23. Söding, J. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960 (2005)

    Article  PubMed  Google Scholar 

  24. 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)

    Article  ADS  CAS  PubMed  Google Scholar 

  25. 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)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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)

    Article  CAS  PubMed  Google Scholar 

  27. Hofmann, K. P. et al. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009)

    Article  CAS  PubMed  Google Scholar 

  28. 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)

    Article  CAS  PubMed  Google Scholar 

  29. 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)

    Article  CAS  PubMed  Google Scholar 

  30. 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)

    Article  CAS  PubMed  Google Scholar 

  31. Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008)

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. 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)

    Article  CAS  PubMed  Google Scholar 

  34. 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)

    Article  CAS  PubMed  Google Scholar 

  35. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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)

    Article  CAS  PubMed  Google Scholar 

  37. 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)

    Article  PubMed  Google Scholar 

  38. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344, 58–64 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Robertson, N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36–44 (2011)

    Article  CAS  PubMed  Google Scholar 

  41. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

    Article  CAS  PubMed  Google Scholar 

  42. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Leslie, A. G. W. & Powell, H. R. Processing diffraction data with Mosflm. Evolving Meth. Macromolecular Crystallogr. 245, 41–51 (2007)

    Article  Google Scholar 

  45. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Andrew S. Doré, Krzysztof Okrasa, Jayesh C. Patel and Maria Serrano-Vega: These authors contributed equally to this work.

Authors and Affiliations

  1. Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK,

    Andrew S. Doré, Krzysztof Okrasa, Jayesh C. Patel, Maria Serrano-Vega, Kirstie Bennett, Robert M. Cooke, James C. Errey, Ali Jazayeri, Samir Khan, Ben Tehan, Malcolm Weir, Giselle R. Wiggin & Fiona H. Marshall

Authors
  1. Andrew S. Doré
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Krzysztof Okrasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jayesh C. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Serrano-Vega
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kirstie Bennett
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert M. Cooke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. James C. Errey
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ali Jazayeri
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Samir Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ben Tehan
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Malcolm Weir
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Giselle R. Wiggin
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Fiona H. Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C.P., G.R.W. and S.K. carried out the conformational thermostabilization of the constructs and determined the stability of the StaR in a panel of reagents/additives to aid purification and crystallization. K.B. carried out the pharmacology. A.J. devised the stabilization strategy and designed the T4L fusion matrix. A.J. and J.C.P. carried out functional analyses the mutants. J.C.E. contributed to construct design and purification strategy. M.S.-V. characterized truncation constructs and established procedures for, and carried out expression and purification. K.O. performed expression, purification, LCP crystallization, optimized purification and performed crystallization in LCP for data collection of the final construct. A.S.D. designed crystallization constructs, established the platform/protocols for, and carried out LCP crystallization and designed crystal optimization, harvested crystals, collected and processed X-ray diffraction data, solved and refined the structure, and devised functional mutations. Computational analysis of the structure and modelling was carried out by B.T. and A.S.D. Project management was carried out by A.J., R.M.C., M.W. and F.H.M. The manuscript was prepared by A.S.D., B.T., A.J., R.M.C., K.B. and F.H.M.

Corresponding author

Correspondence to Fiona H. Marshall.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of wild-type and thermostabilized mGlu5.

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.

Extended Data Figure 2 Pharmacology of mGlu5StaR(569–836)-T4L.

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.

Extended Data Figure 3 Crystal packing in the mGlu5StaR(569–836)-T4L monoclinic C121 system.

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.

Extended Data Figure 4 T4L mediated contacts in the mGlu5StaR(569–836)-T4L monoclinic C121 system.

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.

Extended Data Figure 5 Constitutive activity of mGlu5 mutants.

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.

Extended Data Table 1 Comparison of the pharmacology of mGlu5 and mGlu5 StaR(569–836)-T4L
Full size table
Extended Data Table 2 Data collection and refinement statistics for mGlu5StaR(569–836)-T4L
Full size table
Extended Data Table 3 Representative alignment of 22 human class C receptor sequences (taken from an alignment of 156 class C receptor sequences)
Full size table
Extended Data Table 4 Structural differences between mGlu5, class B and selected class A GPCRs
Full size table
Extended Data Table 5 Alignment and comparison of allosteric binding site residues across the 8 human metabotropic glutamate receptors
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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). https://doi.org/10.1038/nature13396

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13396

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing