Extended Data Fig. 9: Binding of C14^linear to the MRGPRX2.

a, Heatmap of pairing of C14^linear mutants with MRGPRX2 WT and MRGPRX2 alanine scanning mutants. The receptor mutants that did not show significantly decreased EC50 values compared to those of the WT receptor when binding to a specific C14^linear mutant are highlighted by red color. Biochemical characterization indicated that P1 and K3 of C14^linear paired with the hydrophobic residue F170^ECL2 and the negatively charged residues E164^4.60 and D184^5.38 of MRGPRX2, respectively. Whereas F5 of C14^linear interacted with F170^ECL2 and F257^7.31 of MRGPRX2, the F6 mutation of C14^linear functionally coupled with the F239^6.51 and W243^6.55 mutations. b, Effects of different C14^linear mutations on C14^linear induced G_αi–G_γ dissociation in MRGPRX2 overexpressing HEK293 cells. Statistical differences between C14^linear WT and mutations were determined by two-sided one-way ANOVA with Tukey test. P < 0.01; ***, P < 0.001, n.s., no significant difference. Data are from three independent experiments. (n=3). (P < 0.0001,  < 0.0001, 0.0557, 0.0011,  < 0.0001,  < 0.0001, 0.0199, 0.0036, 0.0006, 0.0074, 0.0071,  < 0.0001 from left to right). c, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on C14^linear induced G_αi–G_γ dissociation in MRGPRX2 overexpressing cells. Statistical differences between MRGPRX2 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. **, P < 0.01; ***, P < 0.001, n.s., no significant difference. Data are from three independent experiments (n=3). (P=0.0023, 0.0005, 0.0002,  < 0.0001, 0.0002,  < 0.0001, 0.0034, 0.0029, 0.0450, 0.0168 from left to right). d, Pairing of C14^linear mutants with MRGPRX2 mutants to identify hot spot interactions. We compared the binding capacity of C14^linear mutants with that of the C14^linear WT for MRGPRX2 mutations with significant deficiency in G_αi–G_γ dissociation. For a receptor mutation that had impaired response to C14^linear WT but show little deteriorated response to an C14^linear with a selective alanine substitution, the receptor mutated site and ligand mutated site could be paired with each other to identify potential hot spot interactions (highlight in red). Data are from three independent experiments. e-f, Structural representation and EM density of C14^linear peptide (e) and C14^circular peptide (f). Because the C14^linear–MRGPRX2–G_i complex had higher resolution, unbiased alanine scanning mutagenesis of each residue in C14^linear and the pocket residues of MRGPRX2 was exploited to assign the primary sequence of C14^linear in the EM density. g, Structural model of the interactions between the P1-W7 segment of the C14^linear peptide and MRGPRX2, which were derived by computational simulation. h, RMSD of the seven residues P1-W7 in C14^linear (upper panel) and of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) during triplicate 200 ns MD simulations. i-j, The average RMSD and +/- s.e.m. values of the seven residues P1-W7 in C14^linear (upper panel) and of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) from triplicate 200 ns MD simulations. k, Structural representation and EM density corresponding to S11, S12, C13 and K14 of C14^linear.