The nine different membrane-anchored adenylyl cyclase isoforms (AC1–9) in mammals are stimulated by the heterotrimeric G protein, Gα_s, but their response to Gβγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gβγ and a dimeric form of AC5 that could be involved in its regulation. Gβγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C_1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gβγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gβγ and show reduced conditional activation by Gβγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gβγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

哺乳动物中九种不同的膜锚定腺苷酸环化酶异构体 (AC1-9) 受到异源三聚体 G 蛋白 Gα _s的刺激，但它们对 Gβγ 调节的反应是异构体特异性的。在本研究中，我们报道了与 Gβγ 复合的无配体 AC5 的冷冻电子显微镜结构以及可能参与其调节的 AC5 二聚体形式。 Gβγ 与卷曲螺旋结构域结合，该结构域将 AC 跨膜区域与其催化核心以及已知作为异构体特异性调节中心的区域 (C _1b ) 连接起来。我们证实了 Gβγ 与纯化蛋白和基于细胞的测定的相互作用。与人类家族性运动障碍相关的 AC5 功能获得性突变位于 AC5 与 Gβγ 的界面处，并显示 Gβγ 的条件激活减少，强调了观察到的人类运动功能相互作用的重要性。我们提出了一种分子机制，其中 Gβγ 要么阻止 AC5 二聚化，要么变构调节卷曲螺旋结构域，从而调节催化核心。由于我们对单个 AC 亚型如何独特调节的机制理解有限，因此此类研究可能为亚型特异性药物开发提供新途径。