Structural basis of activation and inhibition of the Ca²⁺/calmodulin-sensitive adenylyl cyclase 8

AbstractMembrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between cAMP pathway and Ca2+signalling. Despite the importance of AC8 in physiology, including cognitive functions and memory, 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 Ca2+/CaM and the stimulatory Gαs protein. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and a 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, allow us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as 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.