Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics

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. A structural proteomics approaches, limited proteolysis, and crosslinking mass spectrometry, allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and G, 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.