![]() ![]() These structures provided the first glimpse into the architecture and auto-inhibitory regulation of a full-length membrane AC. Recently we determined the cryo-EM structure of the full-length bovine AC9 bound to Gαs in an autoinhibited “occluded” state, together with the structure of a C-terminally truncated AC9 (AC9 1250), bound to Gαs, forskolin and MANT-GTP, a non-cyclizable AC inhibitor 1. ![]() The mechanism of forskolin-mediated AC activation was ascribed to its ability to “lock” the two domains closer together, likely inducing a conformational change to reorient C1a and C2a with respect to each other to enhance activation 12, 13, 14. Forskolin and Gαs were required to stabilize the dimer of the isolated AC5 C1 and AC2 C2 domains, as both regulators enhance the affinity between C1a and C2a in the soluble system 12, 13, 14. While forskolin is a non-physiological activator of the membrane ACs, an endogenous molecule that regulates the ACs at the allosteric binding site has not yet been identified. These studies provided the structural basis for the two metal-ion-catalysis of ATP-cAMP conversion, revealed some of the intermediate states of the enzyme, and provided a plausible explanation of enzyme activation by the plant-derived small molecule activator, forskolin 15. Insights into the structure and molecular mechanism of the membrane ACs have been gained by the early X-ray crystallographic studies on the chimeric soluble domain of adenylyl cyclase (AC5 c1/AC2 c2) in complex with Gαs and forskolin 12, 13, 14. Each membrane AC contains two conserved cytosolic catalytic domains and twelve predicted transmembrane (TM) helices, with cytosolic N- and C-termini of varied lengths and regulatory roles 1, 11. The membrane ACs share a conserved predicted domain arrangement, membrane topology and a high degree of sequence similarity, particularly in the catalytic regions of the protein 11. Mutations of several membrane ACs have been linked to genetic diseases, including autosomal deafness 44 (AC1) 7, obesity and type 2 diabetes (AC3) 8, familial dyskinesia with facial myokymia (AC5) 9, or lethal congenital contracture syndrome 8 (AC6) 10. For example, the ACs predominantly expressed in the nervous system, AC1 and AC8, are linked to cognitive processes and pain perception 2, whereas in heart, AC5, AC6, and AC9 are linked to heart disease 2, 6. The nine subtypes of membrane ACs described in mammals, AC1-9, differ in cellular localization, tissue distribution, and physiological functions 5. ![]() The produced cAMP is a key second messenger in many living cells, binding to and regulating a number of downstream effector proteins, and thus modulating a plethora of physiological functions 5. The interaction with Gαs potentiates the ACs ability to convert a molecule of adenosine 5’-triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) 1, 4. Activation of a Gαs-coupled GPCR by an extracellular stimulus, such as a hormone, leads to a cascade of events that include the exchange of GDP bound to the Gαs subunit to GTP, dissociation of the GTP-bound Gαs from the heterotrimeric G protein-GPCR complex, followed by binding of the GTP-bound Gαs to a membrane AC. 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.Īdenylyl cyclases (ACs) play a fundamental role in many G protein-coupled receptor (GPCR) mediated signal transduction pathways 1, 2, 3. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. 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. 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. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. ![]()
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