G-protein-coupled receptors (GPCRs) play pivotal roles in various physiological processes. These receptors are activated to different extents by diverse orthosteric ligands and allosteric modulators. However, the mechanisms underlying these variations in signaling activity by allosteric modulators remain largely elusive. Here, we determine the three-dimensional structure of the μ-opioid receptor (MOR), a class A GPCR, in complex with the G_i protein and an allosteric modulator, BMS-986122, using cryogenic electron microscopy. Our results reveal that BMS-986122 binding induces changes in the map densities corresponding to R167^3.50 and Y254^5.58, key residues in the structural motifs conserved among class A GPCRs. Nuclear magnetic resonance analyses of MOR in the absence of the G_i protein reveal that BMS-986122 binding enhances the formation of the interaction between R167^3.50 and Y254^5.58, thus stabilizing the fully-activated conformation, where the intracellular half of TM6 is outward-shifted to allow for interaction with the G_i protein. These findings illuminate that allosteric modulators like BMS-986122 can potentiate receptor activation through alterations in the conformational dynamics in the core region of GPCRs. Together, our results demonstrate the regulatory mechanisms of GPCRs, providing insights into the rational development of therapeutics targeting GPCRs.

G蛋白偶联受体（GPCR）在各种生理过程中发挥着关键作用。这些受体被不同的正构配体和变构调节剂不同程度地激活。然而，变构调节剂信号活性变化背后的机制在很大程度上仍然难以捉摸。在这里，我们使用低温电子显微镜确定了 μ-阿片受体 (MOR)（一种 A 类 GPCR）与 G _i蛋白和变构调节剂 BMS-986122 的复合物的三维结构。我们的结果表明，BMS-986122 结合诱导对应于 R167 ^3.50和 Y254 ^5.58的图谱密度变化，这些残基是 A 类 GPCR 中保守的结构基序中的关键残基。在没有 G _i蛋白的情况下，MOR 的核磁共振分析表明，BMS-986122 结合增强了 R167 ^3.50和 Y254 ^5.58之间相互作用的形成，从而稳定了完全激活的构象，其中 TM6 的细胞内一半向外-转移以允许与 G _i蛋白相互作用。这些发现表明，BMS-986122 等变构调节剂可以通过改变 GPCR 核心区域的构象动力学来增强受体激活。总之，我们的结果证明了 GPCR 的调节机制，为合理开发针对 GPCR 的疗法提供了见解。