Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1–Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.
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Cryo-EM maps and atomic models for the MT1–Gi complex have been deposited in the Electron Microscopy Data Bank and wwPDB with the following accession numbers: EMD-30627 and PDB 7DB6, respectively. The raw images have been deposited in the Electron Microscopy Public Image Archive, under accession code EMPIAR-10714. Source data are provided with this paper.
The Python code and data used for the contact and r.m.s.d. analysis are available on GitHub (https://github.com/raimondifranc/gpcr-gprotein-3D-comparison/).
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We thank T. Nakane for assistance with the single-particle analysis; Y. Nomura and Y. Sato for assistance with the scFv16 expression and purification; K. Sato, S. Nakano and A. Inoue at Tohoku University for assistance with the cell culture and the cell-based GPCR assays; and the staff scientists at The University of Tokyo’s cryo-EM facility, especially K. Kobayashi, H. Yanagisawa, A. Tsutsumi, M. Kikkawa and R. Danev, for help with cryo-EM data collection. This work was supported by the MEXT Grant-in-Aid for Specially Promoted Research (grant no. 16H06294) to O.N.; by the JST CREST program (no. 20344981) ‘Atomic resolution dynamics of supramolecular complexes essential for cellular functions’ to O.N.; by PRIME (JP19gm5910013) and LEAP (JP20gm0010004) grants from the Japan Agency for Medical Research and Development to A.I.; by the JSPS KAKENHI grant nos. 17K08264 (to A.I.) and 17H05000 and 20H03216 (to T.N.) and by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED under grant numbers JP19am0101115 (support number 1111 to O.N.), 19am0101079 (support number 1819; to O.N.) and JP20am0101095 (to A.I.). F.R. thanks the Italian Ministry of University and Research for funding the Department of Excellence Faculty of Sciences of Scuola Normale Superiore. A.I. thanks the Takeda Science Foundation for funding along with The Uehara Memorial Foundation and Ono Medical Research Foundation. R.S. and T.K. acknowledge the Takeda Science Foundation (19H03428), and T.K. thanks the AMED grant (no. JP18gm0910007).
O.N. is a cofounder and scientific advisor for Curreio. All other authors declare no competing interests.
Peer review information Nature Structural & Molecular Biology thanks Alisa Glukhova, Ralf Jockers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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a, Flow-chart depicting the single particle analysis of the MT1-Gi complex. Fourier Shell Correlation (FSC) curve shows 3.3 Å global resolution with the gold standard criteria (FSC=0.143). Local resolution is indicated on the cryo-EM map. b, A representative micrograph of the MT1-ramelteon-Gαi-Gβ1-Gγ2-scFv16 complex. Similar images (3537 in total) were deposited to the EMPIAR (EMPIAR-10714). c, Representative 2D-averaged images of the MT1-ramelteon-Gαi-Gβ1-Gγ2-scFv16 complex. d, The map versus model FSC curves.
a, Cryo-EM density-map of the MT1-ramelteon-Gαi-Gβ1-Gγ2-scFv16 complex (light blue: MT1, yellow: Gαi, green: Gβ1, purple: Gγ2, red: scFv16). The Fab fragment scFv16 binds and stabilizes the interface of Gαi and Gβ1. b, Top view of MT1-Gi and reported lipid GPCRs, viewed from the extracellular side. Receptors (light blue: MT1-Gi, red: TP, blue: EP3, green: EP4) are shown as cylinder models, and ligands are shown as CPK models. MT1-Gi shows the “lid” structure, which is similar to those in the reported crystal structures of lipid GPCRs.
a–c, NanoBiT-G-protein dissociation assays for the constructs used in the cryo-EM analysis and the MT1 mutants. Concentration–response curves for melatonin-dependent G-protein dissociation signals for MT1. To match the expression of MT1-WT to that of mutants with lower expression, 1:5 volume [WT (1:5)] or 1:10 volume [WT (1:10)] MT1-WT plasmid was used. Asterisks denote an increased volume (2.5-fold) of plasmid transfection. Symbols and error bars represent mean and s.e.m. of the indicated independent numbers of experiments (n = 3–5; see Extended Data Table S1). Note that, in most data points, error bars are smaller than the size of the symbols and thus are not visualized in the panels. The results for the construct used in the cryo-EM analysis (a), the F1965.47A residue that is important in MT1-activation (b), and the mutants to test Gi-selectivity (c) are shown. d, Cell surface expression levels of the MT1-constructs. Cells transiently expressing the FLAG epitope-tagged MT1-constructs were labeled with an anti-FLAG tag antibody, and then with an Alexa 488-conjugated secondary antibody. The fluorescent signals from individual cells were measured by a flow cytometer (See Methods). Data from each experiment are shown as dots with the mean value presented as a bar (n = 3–9). For the multiple comparison analysis, two-way ANOVA followed by the Dunnett’s test (WT (1:1) vs. mutant) was performed: ns, not significantly different, *, P < 0.05, *** P < 0.001. Source data for (a–d) are available online.
a,b, Structural comparison in the sagittal cross section (left panels), the lateral pore (top panels) and the transverse cross section (right panels) between MT1-Gi (light blue) and MT1 alone (crystal structure)14 (green, PDB 6ME2). The cross section and focused view on the lateral pore in MT1-Gi are shown. c, Molecular dynamics (MD) simulations in the presence (left, top) and absence (right, top) of ramelteon. The temporal changes in the distance of the Cα atom between Ala1584.56 and His1955.46 in the presence (left, top) and the absence (right, top) of ramelteon are shown. The transverse cross sections at the ligand-binding site at 200 ns and 400 ns for each simulation in the presence (left, below) and absence (right, below) of ramelteon.
Structural comparison of the G-protein interfaces among the GPCR-Gi and GPCR-Gs complexes. Structures are shown as ribbon models. Residues are shown as stick models. Hydrogen bonds and ionic interactions are indicated by black dashed lines. Focused views at the C-terminus of Gα (top panels) and the hydrophobic pocket of Gα (lower panels) are shown for CB1-Gi23 (a; cyan, PDB 6N4B), CB2-Gi27 (b; yellow, PDB 6KPF), NTS1-Gi C-state22 (c; green, PDB 6OS9), NTS1-Gi NC-state22 (d; brown, PDB 6OSA), D2R-Gi24 (e; yellow, PDB 6VMS), GPR52-miniGs41 (f; green, PDB 6LI3), β2AR-Gs43 (g; red, PDB 6NI3), and A2AR-miniGs42 (h; blue, PDB 6GDG).
The NanoBiT-G-protein dissociation assays for concentration-response curves of G-protein dissociation signals are shown for MT1 (red) and mock (grey), using melatonin (top panels) and ramelteon (bottom panels). Symbols and error bars represent mean and s.e.m. of three independent experiments. Note that, in most data points, error bars are smaller than the size of the symbols and thus are not visualized in the panels. Source data are available online.
Cryo-EM density maps and structural models are shown for selected regions: Ligand-binding pocket, Lateral gate, Toggle switch and PIF core, Na+ site and NPxxY motif, DRY motif, TM6 (lower part) and Gi-interface.
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Okamoto, H.H., Miyauchi, H., Inoue, A. et al. Cryo-EM structure of the human MT1–Gi signaling complex. Nat Struct Mol Biol 28, 694–701 (2021). https://doi.org/10.1038/s41594-021-00634-1