Structural basis of adenylyl cyclase 9 activation

Abstract

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Human adenylyl cyclase 9 is auto-stimulated by its isoform-specific C-terminal domain

“…Although full-length AC9 is resistant to regulation by G proteins, the interfaced helices 6 and 12 of the transmembrane arrays continue into the cytoplasm as the transducing helices and form the coiled-coil ( Qi et al, 2019 ). The transmembrane arrays may function as cell-surface receptors and/or sensors of the lipid composition of the plasma membrane, and changes in the relative positions of helices 6 and 12 may be transmitted to the coiled-coil ( Finkbeiner et al, 2019 ; Qi et al, 2019 , 2022 ; Seth et al, 2020 ). Overall, this scenario appears similar to the activation of soluble guanylyl cyclase by nitric oxide ( Horst et al, 2019 ).…”

“…The auto-inhibitory motif of C2b that occludes AC9 when in complex with Gsα is well delineated ( Pálvölgyi et al, 2018 ; Qi et al, 2019 ). With respect to how C2b might stimulate AC9 activity, indirect evidence points to the forskolin binding pocket ( Tang & Hurley, 1998 ; Qi et al, 2022 ). First, the cryo-electron microscopic map of bovine AC9 shows that the C2b domain is capable of short-distance interactions with residues in the forskolin binding pocket ( Qi et al, 2019 ).…”

“…Because of the inherent variability of transient transfections, each well received a mixture of 5 μM forskolin (LC Labs) and 100 μM rolipram (Insight Biotech) at the end of the recording as a quality control stimulus. This standardization was possible because AC9 is not stimulated by 5 μM forskolin even when stimulated by Gsα ( Baldwin et al, 2019 ; Qi et al, 2019 , 2022 ). As the GloSensor response at high levels of cAMP becomes non-linear and eventually saturates ( Binkowski et al, 2011 ), the concentration–response curves may be flattened at high concentrations of isoproterenol.…”

“…Adenylyl cyclase 9 (AC9) is widely distributed in the body and has been implicated in a number of physiological processes, including cardiac function, body fat mass and body weight, and cancer pathologies and atherosclerosis ( Antoni, 2020 ; Ostrom et al, 2022 ). Partial high-resolution maps of the structure of AC9 obtained by cryo-electron microscopy have been recently published ( Qi et al, 2019 , 2022 ). In brief, the 1,353-residue single polypeptide chain of AC9 forms a tripartite structure.…”

The evolutionary conservation of eukaryotic membrane-bound adenylyl cyclase isoforms

“…The C1b region jointly with the N-terminus of mAC7 was implicated in the regulation by the G 13 pathway ( Jiang et al, 2013 ). The C-terminus of mAC2 has been implicated in regulation by phosphorylation ( Levin and Reed, 1995 ; Böl et al, 1997a ; Böl et al, 1997b ; Pálvölgyi et al, 2018 ) and in mAC9 C2b region is implicated in autoinhibition ( Vercellino et al, 2017 ; Qi et al, 2019 ; Qi et al, 2022 ). In mAC8 C2b has been reported as autoinhibitory and to contribute to the stimulation of the enzyme with Ca 2+ /calmodulin ( Macdougall et al, 2009 ).…”