Extended Data Fig. 8: Binding of PAMP-12 to the MRGPRX2.
From: Structure, function and pharmacology of human itch receptor complexes
a, Pairing of PAMP-12 mutants with MRGPRX2 mutants to identify hot spot interactions. We compared the binding capacity of PAMP-12 mutants with that of the PAMP-12 WT for MRGPRX2 mutations with significant deficiency in Gαi–Gγ dissociation. For a receptor mutation that had impaired response to PAMP-12 WT but show little deteriorated response to an PAMP-12 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. The original data of Figure 3a. b, The EM density corresponding to five residues from F9 to W13 of PAMP-12 and their interaction residues in ligand-binding pocket of MRGPRX2. EM density corresponding to PAMP-12 were highlight in orange. c, Structural representation of seven representative clusters derived by covalent docking of seven residues of PAMP-12 using Autodock4. d: Docking clusters were ranked by numbers in each cluster. The representative conformations in the classification with either the largest number (cluster 1) and the lowest binding energy (cluster 2) were used for MD simulations, respectively. e, The average RMSD value of the seven residues N14-R20 (N14, K15, W16, A17, L18, S19, R20) in PAMP-12 (upper panel) and RMSD of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) during triplicate 200 ns MD simulations. f-h, Structural representations of the interactions surrounding the R10 (g), W13 (h), F9 and W16 (i) of PAMP-12 of MRGPRX2. Hydrogen bonds were showed in red dash. i, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on PAMP-12 induced Gαq–Gγ dissociation in MRGPRX2 overexpressing cells. Statistical differences between MRGPRX2 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001. The curve data from at least three independent measurements are measured as mean ± SEM (n=3). (P < 0.0001, < 0.0001, < 0.0001, < 0.0001, 0.0007, < 0.0001, < 0.0001 from top to bottom).
