Extended Data Fig. 7: The ligand binding mode of SPE-mTAAR9-Gs structure.

The EM densities of SPE and DMCHA could be positioned in 3 different modes, and PEA in 2 potential modes in the SPE-, DMCHA- and PEA-mTAAR9-Gs complex structures. After molecular dynamics (MD) simulations, only 1 mode of PEA, SPE, and DMCHA was used for further structural analysis and biochemical characterizations (also see supplementary Figs. 11–12). However, the CAD binding mode within the mTAAR9 ligand pocket could not be precisely defined, in which multiple conformations of CAD could be fit with current EM density. a, EM density corresponding to the three different modes of SPE in SPE-mTAAR9-Gs complex. SPE-mode 1: green, SPE-mode 2: orange, SPE-mode 3: blue. b, 3D representation of the detailed interactions between SPE and mTAAR9 in mode 1, mode 2 and mode 3. Hydrogen bonds are shown as red dashed lines. c, Barcode representation of interaction patterns in three binding modes of mTAAR9 bound by SPE. Residues that interact with SPE are indicated in green circles, those that interact only in mode 1 or mode 2 are shown in light blue circles, and those that interact only in mode 3 are shown in orange circles. d, The binding energy contribution comparison of binding site residues of mTAAR9 in the two modes of SPE-mTAAR9-Gs complex as calculated by the molecular mechanics MM-PBSA method⁵⁷. SPE-mode 1: green, SPE-mode 2: orange. e, Effects of mutations of interacting residues in three SPE-binding modes of mTAAR9 in response to the stimulation with SPE. The heatmap is colored according to the value of ΔpEC50. Values were from 3 independent experiments (n = 3). Mutations of residues that interacted in mode 1 but not in mode 3 are shown in light blue background, and those that interact only in mode 3 are shown in orange background. f, The average RMSD value of SPE and binding site residues in the three modes of SPE-mTAAR9-Gs complex during triplicate 200 ns MD simulations. Notably, the binding mode 1 and mode 2 of SPE were very similar, except for the N5 of SPE in SPE-mTAAR9-Gs could be positioned differently that all fit well with EM density, potentially due to the linear configuration of SPE. Compared to the other mode (mode 2), the binding mode with SPE (mode 1) exhibited lower energy when the ligand was in proximity to F117^3.37 and the middle N5 was closer to Y274^6.51. Moreover, mutating F117^3.37A significantly decreased mTAAR9 activation in response to SPE stimulation. Further analysis of the binding energy contributions of each residue located in the binding pocket by MD analysis confirmed that binding mode 1 exhibited a greater stability than that of mode 2. In mode 3, the SPE lost specific interactions with the T109^3.29, F168^4.61, W271^6.48 and V300^7.42, and formed new contact with Y293^7.35. Notably, despite Y293A mutation showed no significant effects on SPE induced mTAAR9 activation, mutations of T109^3.29A, F168^4.61A, W271^6.48A or V300^7.42A either significantly reduced or totally abolished SPE induced mTAAR9 activation. Moreover, the MD simulation suggested that mode 3 is less stable than both mode 1 and mode 2. We therefore mainly used mode 1 of SPE for further biochemical characterizations.