XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity

Abstract

The human MT₁ and MT₂ melatonin receptors^1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns³. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer³, and MT₂ has also been implicated in type 2 diabetes^4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT₂ receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon⁶ at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H208^5.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature⁷) and N86^2.50D, obtained in complex with 2-PMT. Comparison of the structures of MT₂ with a published structure⁸ of MT₁ reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [³H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT₁ and MT₂, but in addition the MT₂ structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT₂. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

Extended Data Fig. 1 |. Crystallisation of MT₂: crystals, crystal packing, and electron density.

a, Bright field and b, cross-polarised images of representative MT₂-2-pmt crystals optimized for synchrotron data collection (representing three independent crystallisation setups). c, cross-polarised image of representative MT₂-N86D-2-pmt crystals used for XFEL data collection (representing three independent crystallisation setups). See Extended Data Table 2 for data collection statistics. d, e, Crystal packing (receptor - purple, BRIL – green, and rubredoxin - blue). Space for missing rubredoxin in molecule B of the asymmetric unit is indicated with a red circle. Lattice rotated 90° is shown in e. f, Overlay of 2-pmt (purple) and ramelteon (blue) ligands of MT₂. g-e, 2mF_o-DF_c density (grey) contoured at 1 σ of ramelteon (g), N86^2.50D mutation (h), and H208^5.46A mutation (i). 2-pmt is shown in purple.

Extended Data Fig. 2 |. Structural differences between MT₁ and MT₂.

a, Overlay between MT₁-2-pmt (green) and MT₂-2-pmt (violet) structures (Cα r.m.s.d = 0.6 Å). b, Comparison of MT₁ (green) and MT₂ (violet) binding pockets. Overall, the binding pocket is about 50 ų larger for MT₂. c, Comparison of 2-pmt ligand conformations in MT₁ (green) and MT₂ (violet). Hydrogen bonds are shown as yellow dashed lines. d, Overlay of MT₁ and MT₂, showing residues that display different conformations in the vicinity of the binding pocket. N^4.60 makes a hydrogen bond with Y^5.38 in MT₂ but not in MT₁.

Extended Data Fig. 3 |. Molecular dynamics simulations.

a, b, Distance plots for interactions between residues in MT₂ (N175^4.60, atom type ND2 (N^δ); Q194^ECL2, atom NE2 (N^ε); N268^6.52, atom ND2), and closest oxygen atoms of the ligand methoxy and acetyl groups, respectively, in complexes with melatonin (a) and 2-pmt (b) from three independent simulations runs. c, Distance histograms for interactions of methoxy with N175^4.60 with melatonin (yellow) and 2-pmt (violet). d, Distance histograms for interactions of methoxy with and Q194^ECL2 with ligand alkylamide tail with melatonin (yellow) and 2-pmt (violet).

Extended Data Fig. 4 |. Structural and functional differences between MT₂-pmt and MT₂-H208A^5.46-2-pmt.

a, Overlay of the MT₂-2-pmt (purple) structure with MT₂-H208^5.46A-2-pmt (grey) reveals an inward shift of helix V of ~0.9 Å due to the H208^5.46A mutation (as shown by black arrow). b, Surface representation of the H208^5.46 and H208^5.46A residues. Rotation of helix V renders the binding pocket volume ~50 ų smaller for the H208^5.46A structure (binding site volume for MT₂-2-pmt: 766 ų compared to 716 ų for the MT₂-H208^5.46A structure). c, Comparison of the channel profiles (from the outside of the protein towards the ligand) for MT₂-2-pmt (purple) and MT₂-H208^5.46A-2-pmt (grey) reveals a narrowing of the MT₂-H208^5.46A-2-pmt channel around 6 Å as a consequence of the mutation and subsequent inward rotation of helix V. d, Functional data for WT and the H208^5.46A mutant expressed in HEK293T cells by using GloSensor to measure Gi/o-mediated cAMP inhibition. Data represent mean ± s.e.m. for n independent experiments as indicated in square brackets. %E_MAX is relative to wild-type receptor (in columns), and (%E_MAX*) is relative to melatonin activity (in rows). See Methods for further information and Supplementary Figure 6 for dose response curves.

Extended Data Fig. 5 |. Selectivity analysis of melatonergic compounds.

a, Binding affinities of ligands for MT₁ (ChEMBL target identifier CHEMBL1945) and MT₂ (CHEMBL1946) were retrieved from the ChEMBL database (v. 24) of experimental literature values. Of these ligands, 525 have affinities reported for both receptor subtypes. For ligands with multiple reported affinity values for a given receptor, pK_i values were averaged. MT₁-selective ligands are in the lower right quadrant; MT₂-selective ligands are in the upper left quadrant. Data points are coloured by absolute pK_i difference between subtypes, i.e. selectivity. b, Histogram of observed ligand selectivities. MT₂ selective ligands are on the left of the panel, MT₁ selective ligands are on the right. c, Plot of the docking score difference of select ligands that were docked between MT₂ and MT₁ versus their pK_i difference (MT₂-MT₁). Dashed lines indicate pK_i selectivity cutoff criteria (MT₁: 1 and MT₂: −4). Data points are colored by molecular weight (Da). See Supplementary Table 1 for details of docked ligands.

Fig. 1 |. Overview of the MT₂ structure.

a, Overview of MT₂ (violet) shows the canonical 7TM topology, with the ligand 2-pmt (purple) in the binding pocket. A 90° view shows the receptor from the extracellular side. Approximate membrane boundaries are shown as grey lines. b, 2mF_o-DF_c density (grey mesh) of 2-pmt contoured at 1 σ. c, Binding pocket with key ligand interaction residues. d, Schematic diagram of ligand-interacting residues. Residues in the hydrophobic sub-pocket are coloured green. Hydrogen bonds are shown as dashed yellow lines in c and d.

Fig. 2 |. Two possible ligand entries in MT₂.

a, View of the membrane-buried channel in MT₂. Insert shows the channel diameter profile across its length for MT₁ and MT₂. b, A 90° view of the channel in MT₂, highlighting three residues discussed in the text. c, The same as in b view of MT₁ (green) showing a different conformation of Y187^5.38 that widens the channel compared to MT₂. d, View of the ECL opening found in MT₂ (violet) with 2-pmt (purple). Insert shows the ECL opening profile across the length. e, A 90° view through the ECL opening in MT₂, highlighting three residues discussed in the text. f, The same as in e view of MT₁ (green), showing a different conformation of Y281^7.39 that seals the ECL opening. g, [³H]-melatonin dissociation kinetics for MT₂ membrane channel mutants (top) and ECL opening mutants (bottom). h, same as in g for MT_1. Residence time (1/k_off) in g and h is given in minutes. Data are shown as mean±s.e.m. for n=3 independent experiments.

Fig. 3 |. Selectivity determinants of ligands at MT₁ and MT₂.

a, Docking of selective ligands into MT₁ (green), with 2-pmt (purple) from the crystal structure shown as reference. Ligands selective for MT₁ (compounds 63, 64, 65a, and 65b) are shown in grey. Two representative ligands, 5-HEAT and CTL 01–05-B-A05⁸ are coloured pale yellow, with their selectivity-conferring substituents (R¹ position) shown in orange. b, Docking of ligands into MT₂ (violet), with 2-pmt (purple) shown as reference. Non-selective (tasimelteon, TIK301²²) and selective (UCM1014, K185, and 4P-PDOT) ligands are shown in grey. Two representative ligands, DH97¹⁷ and IIK7¹⁷ are coloured pale yellow, with selectivity-conferring substituents (R² and R³ positions) shown in cyan. Predicted hydrogen bonds are shown as dotted lines in a and b. c, Melatonin SAR, where R¹ substituents confer MT₁ selectivity (orange), and substituents in R² and R³ positions confer MT₂ selectivity (cyan). See Supplementary Table 1 for a list of all docked ligands.

Fig. 4 |. MT₂ mutations implicated in type 2 diabetes.

Mapping of residues implicated in T2D as described in Refs.^, on the MT₂ crystal structure. Residues, mutations of which lead to defects in two or more pathways, are coloured gold, G protein-specific defects - cyan, β-arrestin 2-specific - blue, ERK-specific – magenta, mutations abolishing melatonin-binding are shown in green, and those similar to WT shown in grey. T2D mutations in residues, not observed in the crystal structure, are not shown.