PDF(Pubmed)
Abstract：
Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
摘要：
膜腺苷酸环化酶AC8受G蛋白和钙调蛋白（CaM）调节，介导cAMP通路和Ca2+信号之间的串扰。尽管AC8在生理学中很重要，G蛋白和CaM调节的结构基础尚不明确。这里,我们报告了在Ca2/CaM存在下与刺激性Gαs蛋白结合的牛AC8的3.5µ分辨率低温EM结构。该结构揭示了与Gαs和小分子激活剂毛喉素结合的有序AC8结构域的结构。AC8的细胞外表面具有带负电荷的口袋，未知交互者的潜在站点。尽管毛喉素密度很好，AC8的捕获状态不利于紧密的核苷酸结合。结构蛋白质组学方法，有限的蛋白水解和交联质谱（LiP-MS和XL-MS），允许我们确定AC8及其监管机构之间的联系点，CaM,Gαs,和Gβγ，以及推断这些相互作用引起的构象变化。我们的结果为理解灵活区域在AC调节机制中的作用提供了框架。
