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), operating at a crossroads between the GPCR/cAMP pathway and Ca²⁺ signalling pathway. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. The Korkhov group (Institute of Molecular Biology and Biophysics, ETH Zurich & Paul Scherrer Institute), in collaboration with the groups of Paola Picotti and Alexander Leitner (Institute of Molecular Systems Biology, ETH Zurich), determined the cryo-EM structure of the bovine AC8 bound the stimulatory Gαs protein in the presence of Ca²⁺/CaM. The structure revealed 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 interaction partners.

While the structure revealed a well resolved density corresponding to the activating small molecule, forskolin, the state of AC8 captured by cryo-EM does not appear to favour tight nucleotide binding. The authors combined the cryo-EM analysis of the AC8 protein complex with the mass spectrometry-based structural proteomics approaches: limited proteolysis and crosslinking mass spectrometry. This integrative structural biology approach allowed them 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. The results of this study, published in EMBO Reports, provide a framework for understanding the role of flexible regions in the mechanism of adenylyl cyclase regulation.

Link to the paper in external page "EMBO Reports".­