Extended Data Fig. 2. The binding pharmacology of momSalB and GR89,696.

a. GPCRome screening at 320 GPCRs that measures agonist activity of tested ligands shows that momSalB and GR89,696 are selective at KOR. b. The binding poses of momSalB and GR89,696 show that they adopt different planes in the orthosteric pocket. c. A cartoon model of G protein-mediated cAMP reporter assays. The Gαi/o here represents all subtypes expressed in the cells. d. Mutagenesis screening of key binding-pocket residues using G protein-mediated cAMP reporter assay. Data are grouped data ± s.e.m. of n = 3 biological replicates. Full quantitative parameters from this experiment are listed in Supplementary Table 2. e. Measurement of cell surface expression of KOR binding pocket mutants by ELISA. In general, these mutants maintained robust cell surface expression. Although some mutations significantly altered surface expression compared to the wild type, the increased or decreased expression appeared to minimally affect the potency in agonist-mediated cAMP inhibition. Bar-graphs are OD₄₅₀ ± s.e.m. from n = 3 biological replicates. Statistical significance for each mutant is compared in a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test to the wild type (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, “ns” represents no significance; V108A: p = 0.3919, W124A: p = 0.0893, V134A: p = 0.0308, M142A: p = 0.0398, V230A: p = 0.0004, H291A: p = 0.0002, Y320L: p = 0.0008). Signalling curves are grouped data ± s.e.m. of n = 3 biological replicates. Full quantitative parameters from this experiment are listed in Supplementary Table 9.