Allosteric modulators of G-protein-coupled receptors (GPCRs) enhance signaling by binding to GPCRs concurrently with their orthosteric ligands, offering a novel approach to overcome the efficacy limitations of conventional orthosteric ligands. However, the structural mechanism by which allosteric modulators mediate GPCR signaling remains largely unknown. Here, to elucidate the mechanism of μ-opioid receptor (MOR) activation by allosteric modulators, we conducted solution NMR analyses of MOR by monitoring the signals from methionine methyl groups. We found that the intracellular side of MOR exists in an equilibrium between three conformations with different activities. Interestingly, the populations in the equilibrium determine the apparent signaling activity of MOR. Our analyses also revealed that the equilibrium is not fully shifted to the conformation with the highest activity even in the full agonist-bound state, where the intracellular half of TM6 is outward-shifted. Surprisingly, an allosteric modulator for MOR, BMS-986122, shifted the equilibrium toward the conformation with the highest activity, leading to the increased activity of MOR in the full agonist-bound state. We also determined that BMS-986122 binds to a cleft in the transmembrane region around T162 on TM3. Together, these results suggest that BMS-986122 binding to TM3 increases the activity of MOR by rearranging the direct interactions of TM3 and TM6, thus stabilizing TM6 in the outward-shifted position which is favorable for G-protein binding. These findings shed light on the rational developments of novel allosteric modulators that activate GPCRs further than orthosteric ligands alone and pave the way for next-generation GPCR-targeting therapeutics.

中文翻译：

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变构调节剂激活μ-阿片受体的机制。

G 蛋白偶联受体 (GPCR) 的变构调节剂通过与其正构配体同时与 GPCR 结合来增强信号传导，为克服传统正构配体的功效限制提供了一种新方法。然而，变构调节剂介导 GPCR 信号传导的结构机制仍然很大程度上未知。在这里，为了阐明变构调节剂激活 μ-阿片受体 (MOR) 的机制，我们通过监测来自甲硫氨酸甲基的信号对 MOR 进行了溶液 NMR 分析。我们发现MOR的细胞内侧存在于具有不同活性的三种构象之间的平衡。有趣的是，平衡中的种群决定了 MOR 的明显信号活性。我们的分析还表明，即使在完全激动剂结合状态下，平衡也没有完全转变为具有最高活性的构象，其中 TM6 的细胞内一半向外移动。令人惊讶的是，MOR 的变构调节剂 BMS-986122 将平衡转移到具有最高活性的构象，导致在完全激动剂结合状态下 MOR 的活性增加。我们还确定 BMS-986122 与 TM3 上 T162 周围的跨膜区域中的裂缝结合。总之，这些结果表明，BMS-986122 与 TM3 的结合通过重新排列 TM3 和 TM6 的直接相互作用来增加 MOR 的活性，从而将 TM6 稳定在有利于 G 蛋白结合的向外移位的位置。