Extended Data Fig. 11: Active state and G protein coupling of MRGPRX2.

a, Structural comparison of TM3 and TM6 in the MRGPRX2–G_i1 complex (green), CB1–G_i complex (cyan, PDB: 6N4B) and μ-opioid–G_i complex (blue, PDB: 6N4B). Compared to the corresponding structures in the CB1 and μ-opioid receptors, the upper TM6 of MRGPRX2 is distorted inwardly at the position of G236^6.48 in MRGPRX2. b, Structural comparison of residues involved in kink formation in MRGPRX2 (green), CB1 (cyan, PDB: 6N4B) and μ-opioid receptor (blue, PDB: 6N4B). The engagement of the phenolic oxygen of Y113^3.36 and the main chain of G236^6.48 in MRGPRX2 at the kink position was further stabilized by packing between F239^6.51 and Y113^3.36. Toggle switch W^6.48 of the CB1, ADGRG3 and µ-opioid receptors was replaced by G236^6.48 in MRGPRX2. H-bonds are depicted as dashed lines. c, Structural comparison of TM6 in MRGPRX2 (green), CB1 (cyan, PDB: 6N4B), μ-opioid receptor (light blue, PDB: 6N4B), ADGRG3 (yellow orange, PDB: 7D76), GLPR1 (blue, PDB: 6B3J) and GABA1 (grey, PDB: 7C7Q). d, Cutaway view of G^6.48XXF^6.52 motif in C14^linear–MRGPRX2–G_i1 and C48/80–MRGPRX2–G_i1 complex. Hydrogen bonds are depicted as red dashed lines. e, Schematic representation of the FlAsH-BRET assay. The Nluc was inserted at the P102 position close to extracellular end of TM3 and the FlAsH motif were inserted at the K251 position of the extracellular end of TM6 (upper panel). Representative dose response curves of the conformational changes in response to binding of C 48/80 or Compound 2 reported by FlAsH-BRET assay. A future solution of the inactive structure of MRGPRX2 and comparison with our active structure will provide further evidence of whether kink formation of TM6 is required for the activation of MRGPRX2. Data from three independent experiments are presented as the mean ± SEM (n=3) (lower panel). f, Comparison of the D/ERY motif and NPXXY motif in MRGPRX2–G_i (green), CB1–G_i (gray) and μ-opioid (yellow) complex, which showed similar conformations. g, Structural representation of the MRGPRX2–G_i1 complex (green). TM3-TM5 and ICL1-ICL3 of MRGPRX2 and the αN-helix, α5-helix, i3 loop, and β2-β3 loop of G_αi are highlighted. h, Comparison of the G_i coupling interfaces in cryo-EM structures of MRGPRX2–G_i1 complexes, the μ-opioid–G_i complex (PDB: 6N4B), the CB1–G_i complex (PDB: 6N4B), and the model of MRGPRX2–G_αq generated by computational simulation. Residues of MRGPRX2, μ-opioid receptor or CB1 in contact with G_i are illustrated as green or blue dots. The basic charged R that contributes to the interface is highlighted in blue. Residues that contact G_αi but not G_αq are illustrated as brown dots. i, Detailed interactions between ICL1 and ICL3 of MRGPRX2 and the G_i1 trimer. R60^ICL1 inserted in a hydrophobic pocket created by L55 and F335 and formed a hydrogen bond with S334 of the G_β1 subunit, R214 ^ICL3 formed a cation-π interaction with Y320 and potential charge interactions with E318 of G_αi1.