Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.

鉴于 G 蛋白偶联受体 (GPCRs) 的变构调节剂具有良好的临床价值，对这些调节剂如何改变 GPCR 功能的机械理解具有重要意义。在这里，我们报告了与正变构调节剂 (PAM) ZCZ011 结合的大麻素受体 CB1 的晶体学和低温电子显微镜结构。这些结构表明 ZCZ011 与跨膜 2 (TM2)-TM3-TM4 表面的螺旋外位点结合。通过（非）偏向分子动力学模拟和诱变实验，我们表明 TM2 重排对于变构信号的传播至关重要。ZCZ011 通过促进 TM2 重排有利于受体激活和增加采用活性构象的受体数量来发挥 PAM 效应。相比之下，ORG27569，CB1 的负变构调节剂 (NAM)，也与 TM2-TM3-TM4 表面结合并通过阻止 TM2 重排发挥 NAM 效应。我们的研究结果填补了对 CB1 变构调节的理解的空白，并可以指导 CB1 变构调节剂的合理设计。