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. 2023 May 3;617(7960):417–425. doi: 10.1038/s41586-023-06030-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended data Fig. 5 — a. Mutagenesis screening of KOR-G protein interface (KOR side) residues by cAMP inhibition assays. Data are grouped data ± s.e.m. from n = 3 biological replicates. b. Mutagenesis analysis of intracellular KOR residues by cAMP inhibition assays. Data are mean LogEC50 ± 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, GR89,696: V160A: p = 0.0001, P163A: p = 0.0020, R252A: p = 0.0014, L253A: p = 0.0003, R257A: p = 0.0003, R271A: p = 0.0004; momSalB: L167A: p = 0.0003, R257A: p = 0.1514, R271A: p = 0.0004, I272A: p = 0.0002). Full quantitative parameters from this experiment are listed in Supplementary Table 12. c. Measurement of cell surface expression of KOR-G protein interface mutants by ELISA. The BRET results suggest a minor effect with different concentrations of KOR plasmids. Bar-graphs are OD₄₅₀ ± s.e.m. from n = 3 independent biological replicates. Statistical significance for each mutant is compared in a one-way analysis of variance (ANOVA) with the 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; V160A: p = 0.2732, P163A: p = 0.9992, R252A: p = 0.9998, L253A: p = 0.8943, R257A: p = 0.9997, N336A: p = 0.9992). Signalling curves are grouped data ± s.e.m. of n = 3 biological replicates. Full quantitative parameters from this experiment are listed in Supplementary Table 13. d. The KOR-R156A mediated decrease of efficacy was confirmed by the BRET2-based kinetic measurement. Data are net BRET ± s.e.m. from n = 3 independent biological replicates.