• Authors: Okamoto HH. et al.
  • Year: 2021
  • Journal: Nat Struct Mol Biol 28 694-701
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293S


MT1 receptor variants were expressed in HEK-293S GNTI- cells using FectoPRO and grown at 37°C for 16h. The transfected cells were incubated in FreeStyle 293 Expression Medium with 2% FBS at 37°C for 16h. After sodium butyrate was added to 10 nM, the cells were incubated further at 30°C for 48h. Cells were then lysed by osmotic shock using hypotonic buffer.


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.