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Structural basis of ligand recognition at the human MT1 melatonin receptor

Nature volume 569, pages 284–288 (2019)Cite this article

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A Publisher Correction to this article was published on 03 May 2019

This article has been updated

Abstract

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

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Fig. 1: Structural features of MT1.
Fig. 2: Ligand recognition at MT1.
Fig. 3: Docking model of bitopic ligand.
Fig. 4: Comparison between MT1 and 5-HT2C.

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Data availability

Coordinates and structure factors were deposited in the Protein Data Bank (PDB) under the following accession codes: 6ME2 (MT1-CC–ramelteon), 6ME3 (MT1-CC–2-PMT), 6ME4 (MT1-CC–2-iodomelatonin), and 6ME5 (MT1-CC–agomelatine).

Change history

  • 03 May 2019

    Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

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Acknowledgements

We thank M. Chu, C. Hanson, K. Villers, J. Velasquez, and H. Shaye for technical support, and D.R. Mende for useful discussion of sequence analysis. This research was supported by the National Institutes of Health (NIH) grants R35 GM127086 (V.C.), R21 DA042298 (W.L.), R01 GM124152 (W.L.), R01 MH112205 (B.L.R.), and U24DK116195 (B.L.R.), the NIMH Psychoactive Drug Screening Program contract (B.L.R.), F31-NS093917 (R.H.J.O.), the National Science Foundation (NSF) BioXFEL Science and Technology Center 1231306 (B.S., W.L., U.W., T.D.G., V.C.), EMBO ALTF 677-2014 (B.S.), HFSP long-term fellowship LT000046/2014-L (L.C.J.), and a postdoctoral fellowship from the Swedish Research Council (L.C.J.). C.G. thanks the SLAC National Accelerator Laboratory and the Department of Energy for financial support through the Panofsky fellowship. T.A.W. and W.B. acknowledge financial support from the Helmholtz Association via Programme-Oriented Funding. Parts of this research were carried out at the LCLS, a National User Facility operated by Stanford University on behalf of the US Department of Energy and supported by the US Department of Energy Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515.

Reviewer information

Nature thanks Christian Siebold, Ieva Sutkeviciute, Jean-Pierre Vilardaga and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. John D. McCorvy

    Present address: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA

  2. These authors contributed equally: Benjamin Stauch, Linda C. Johansson

Authors and Affiliations

  1. Bridge Institute,USC Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA

    Benjamin Stauch, Linda C. Johansson, Nilkanth Patel, Gye Won Han, Andrii Ishchenko, Nairie Michaelian, Jessica M. Grandner, Anna Shiriaeva, Raymond C. Stevens, Vsevolod Katritch & Vadim Cherezov

  2. Department of Chemistry, University of Southern California, Los Angeles, CA, USA

    Benjamin Stauch, Linda C. Johansson, Gye Won Han, Andrii Ishchenko, Nairie Michaelian, Anna Shiriaeva, Raymond C. Stevens, Vsevolod Katritch & Vadim Cherezov

  3. Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA

    Nilkanth Patel, Jessica M. Grandner, Raymond C. Stevens, Vsevolod Katritch & Vadim Cherezov

  4. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    John D. McCorvy, Xi-Ping Huang, Samuel T. Slocum, Reid H. J. Olsen, Alexandra R. Tribo & Bryan L. Roth

  5. National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Xi-Ping Huang & Bryan L. Roth

  6. SLAC National Accelerator Laboratory, Bioscience Division, Menlo Park, CA, USA

    Cornelius Gati

  7. Stanford University, Department of Structural Biology, Stanford, CA, USA

    Cornelius Gati

  8. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    Alexander Batyuk

  9. Center for Free-Electron Laser Science, DESY, Hamburg, Germany

    Wolfgang Brehm & Thomas A. White

  10. Department of Physics, Arizona State University, Tempe, AZ, USA

    Caleb Madsen & Uwe Weierstall

  11. School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ, USA

    Lan Zhu, Uwe Weierstall & Wei Liu

  12. Hauptman-Woodward Institute, Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA

    Thomas D. Grant

  13. Univ Lille, Inserm, CHU Lille, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Lille, France

    Saïd Yous

  14. Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Bryan L. Roth

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B.S., L.C.J., W.L. and V.C. conceived the project, analysed data and wrote the manuscript with contributions from all authors. B.S. and L.C.J. cloned and characterized the receptor, generated all constructs, crystallized the receptor, prepared all crystal samples and figures, solved and refined the structures, and assisted in generating mutant constructs for binding and functional analyses. B.S. designed thermostabilizing point mutations and performed sequence analysis. J.D.M., X.-P.H. and S.T.S. performed radioligand binding and functional experiments, assisted in generating mutant and wild-type constructs used for binding and functional analyses, and analysed all binding and functional data. A.I., N.M., A.S., L.Z. and W.L. assisted in XFEL sample preparation. G.W.H. performed structure refinement and quality control. B.S., L.C.J., A.B., L.Z., W.L. and V.C. collected XFEL data. C.G., W.B., T.A.W. and T.D.G. processed XFEL data and solved the indexing ambiguity. C.M. and U.W. operated the LCP injector during XFEL data collection. N.P. performed molecular docking and molecular dynamics calculations and assisted in preparing figures. J.M.G. assisted in docking calculations. V.K. supervised molecular docking and molecular dynamics calculations. R.H.J.O. assisted with molecular biology and generating mutant constructs. A.R.T. assisted with generating mutant constructs and functional experiments. S.Y. synthesized the bitopic compound, analysed data and edited the paper. R.C.S. contributed to study design and selection of chemical compounds for receptor stabilization and functional characterization, supervised protein expression and edited the paper. B.L.R. supervised pharmacological experiments and edited the paper. W.L. supervised the LCP crystallization and optimization experiments. V.C. coordinated and supervised the whole project.

Corresponding authors

Correspondence to Bryan L. Roth, Wei Liu or Vadim Cherezov.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Crystals, ligand electron density maps, and packing of MT1.

a, b, Bright field (a) and cross-polarized (b) images of representative MT1–2-PMT crystals, optimized for synchrotron data collection (representing three independent crystallization setups). c, Cross-polarized image of representative MT1–ramelteon crystals used for XFEL data collection (representing two independent crystallization setups). d, 2mFo − DFc ligand electron density maps of MT1 co-crystallized with 2-PMT (orange), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 1.0σ (grey mesh). e, 2mFo − DFc (blue, contoured at 1.0σ) and mFo − DFc (green/red, ±3.5σ) electron density maps of MT1–ramelteon (ligand purple, protein yellow) illustrating the small, unassigned electron density close to N2556.52 that is tentatively attributed to the essential additive 2-propan-ol. The distance from this electron density to the closest ligand atom is approximately 4.8 Å. f, Packing of MT1–PGS crystallized in the P4 21 2 space group. The receptor is shown in green and the PGS fusion protein is shown in purple. g, Simulated annealing mFo − DFc omit maps (green mesh) of 2-PMT (orange sticks), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 3.0σ.

Extended Data Fig. 2 Molecular dynamics simulations.

a, b, Distance plots for interactions between residues in MT1 (N1624.60, atom type ND2 (Nδ); Q181ECL2, atom NE2 (Nε); N2556.52, atom ND2), and the closest oxygen atoms of the methoxy and acetyl groups, respectively, in the ligands melatonin (a) and 2-PMT (b) from three independent simulation runs. c, Distance histograms for interactions of methoxy with N1624.60 (left), and Q181ECL2 with the ligand acetyl tail (right), in melatonin and 2-PMT complexes. d, Hydration of residue N2556.52 over the course of a 1-µs simulation of the MT1–2-PMT complex from three independent simulations. e, Stability of ligand binding in simulations of MT1 complexes. Time dependence of r.m.s.d. for non-hydrogen atoms of melatonin shown for MT1–melatonin complex (left) and MT1–2-PMT complex (right). Three independent simulations of crystal construct (purple, blue, light blue) and crystal construct with N2556.52A mutation (orange, light orange, yellow) are shown, spanning 1.5 μs of cumulative time per system. Sampling rate was 10 frames per ns, and solid lines represent moving average values from 50 frames in all cases.

Extended Data Table 1 MT1 radioligand affinity
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Extended Data Table 2 Functional data (Gi/o Glosensor) for crystallogenic mutants
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Extended Data Table 3 MT1 crystallographic data collection and refinement statistics
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Extended Data Table 4 Volumes of enclosed binding sites of class A GPCRs
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Extended Data Table 5 Thermostability data
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Extended Data Table 6 Functional data (Gi/o GloSensor) for mutants of the YPYP motif and the ligand binding site
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Extended Data Table 7 Functional data (Gi/o GloSensor) for mutants of the lateral channel
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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 and an Experimental Section describing synthesis of the bitopic ligand CTL 01-05-B-A05.

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Stauch, B., Johansson, L.C., McCorvy, J.D. et al. Structural basis of ligand recognition at the human MT1 melatonin receptor. Nature 569, 284–288 (2019). https://doi.org/10.1038/s41586-019-1141-3

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