Structural basis of ligand recognition at the human MT1 melatonin receptor

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 aids^8,9, 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 agomelatine^12,13. 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.

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

a, Bright field, and b, cross-polarised images of representative MT₁-2-pmt crystals, optimised for synchrotron data collection (representing three independent crystallization setups). c, Cross-polarised image of representative MT₁-ramelteon crystals used for XFEL data collection (representing two independent crystallization setups). d, 2mF_o−DF_c ligand electron density maps of MT₁ co-crystallised with 2-pmt (orange), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 1.0 σ (grey mesh). e, 2mF_o−DF_c (blue, contoured at 1.0 σ) and mF_o−DF_c (green/red, +/−3.5 σ) electron density maps of MT₁-ramelteon (ligand purple, protein yellow) illustrating the small, unassigned electron density close to N255^6.52 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 crystallised in the P 4 2₁ 2 space group. The receptor is shown in green and the PGS fusion protein is shown in purple. g, Simulated annealing mF_o−DF_c omit maps (green mesh) of 2-pmt (orange sticks), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 3.0 σ.

Extended Data Fig. 2 |. Molecular dynamics simulations.

a, b, Distance plots for interactions between residues in MT₁ (N162^4.60, atom type ND2 (N^δ); Q181^ECL2, atom NE2 (N^ε); N255^6.52, atom ND2), and closest oxygen atoms of the methoxy and acetyl groups, respectively, in ligands melatonin (a) and 2-pmt (b) from three independent simulation runs. c, Distance histograms for interactions of methoxy with N162 (left), and Q181 with ligand acetyl tail (right), in melatonin and 2-pmt complexes. d, Hydration of residue N255^6.52 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 RMSD 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 N255^6.52A 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.

Fig. 1 |. Structural features of MT₁.

a, Overall architecture of MT₁ (green; disulfide yellow, helices labeled with Roman numerals) bound to ramelteon (purple). ICL3 was replaced by a fusion protein and is shown as a dashed line. Approximate boundaries of the hydrophobic slab corresponding to the lipid tails are derived from MD simulation and indicated by orange lines (yellow shaded areas represent s.d.). ECL2 closes off the binding site to the extracellular space. b, Section through the receptor illustrating the lateral ligand access channel. c, Details of the unique YPYP motif in helix II that forms a bulge in proximity to the ligand. Residues are shown as green sticks, and hydrogen bonds as dashed lines. d, Proximity matrix of pore-lining residues, and minimum diameter profile across the length of the channel, calculated using spherical probes.

Fig. 2 |. Ligand recognition at MT₁.

a, Ramelteon (purple) forms specific interactions with side chains of N162 and Q181 of MT₁ (green), and stacks with F179 (all side chains shown as green sticks). b, Chemical structures of melatonergic ligands that were co-crystallised with MT₁ in this work. c, Binding site composition and interactions of ramelteon as a representative for all four complexes. The hydrophobic sub-pocket accommodating substituents at the 2 position of indole-like ligands is shown in green. d, Structure-guided sequence alignment of MT₁ (residue numbering above), MT₂, and GPR50 (residue numbering below) binding site residues. Ligand-interacting residues in MT₁ and MT₂ are highlighted in bold, and Ballesteros-Weinstein residue numbering is provided for reference. e, 2mF_o−DF_c electron density map (grey mesh) in the binding site of the MT₁-ramelteon complex, contoured at 1.0 σ, and simulated annealing mF_o−DF_c omit map (green mesh), contoured at 3.0 σ. Electron density maps for other ligands are shown in Extended Data Fig. 1. f, Overlay of experimental ligand conformations of ramelteon (purple), 2-pmt (orange), 2-iodomelatonin (yellow), and agomelatine (cyan) after receptor superimposition. The conformations of receptor side chains in the binding site are very similar between the complexes and are omitted for clarity.

Fig. 3 |. Docking model of bitopic ligand.

a, Section through MT₁ (grey) showing the best docked pose of the bitopic ligand CTL 01–05-B-A05 (spheres with slate blue carbons). b, Details of receptor ligand interactions. Dashed lines represent hydrogen bonds between ligand moieties and receptor residues N162^4.60, Q181^ECL2, and H195^5.46, respectively. c, Chemical structure of CTL 01–05-B-A05. The ligand protrudes from the lateral channel between helices IV and V. Its core shows minor displacement compared to the experimentally determined conformation of agomelatine (cyan sticks), and it forms favourable interactions with several residues (white sticks) in the periphery of the channel.

Fig. 4 |. Comparison between MT₁ and 5-HT_2C.

a, Crystal structure of agomelatine (cyan sticks, pK_i(MT₁)=8.8) bound to MT₁ (green cartoon). Important residues in the binding site are shown as sticks with green carbons. b, Crystal structure of ergotamine (grey sticks) bound to 5-HT_2C (orange cartoon), and docking model of agomelatine (cyan sticks, pK_i(5-HT_2C)¹³=6.2). c, Chemical structures of melatonin, serotonin, and ergotamine with the substructure shared between serotonin and ergotamine highlighted (magenta). d, Structure-guided sequence alignment in the binding sites of MT₁ and 5-HT_2C receptors with reference sequence numbering (above / below), and Ballesteros-Weinstein numbering (bottom). Residues participating in tight ligand interactions in the respective structure are bold.