Structural Basis of Adenylyl Cyclase 9 Activation

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 G alpha s subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-G alpha s 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 ATP alpha S, (iv) bound to G alpha s and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the G alpha s 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.