Structural basis of adenylyl cyclase 9 activation

Chao Qi,Basavraj Khanppnavar,Volodymyr M Korkhov,Carmen W Dessauer,Birgit Dreier,Andreas Plückthun,Inayathulla Mohammed,Pia Lavriha,Jonas V Schaefer,Yong Li,Michalis Lazaratos,Ana-Nicoleta Bondar,Ved Mehta

doi:10.1038/s41467-022-28685-y

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.

Highlights

6, Ana-Nicoleta Bondar[5,7], Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts adenosine 5’-triphosphate (ATP) into cyclic adenosine monophosphate (cAMP)
Our choice of MANT-GTP as a ligand for the active site of AC9 was based on two considerations: (i) we have used it successfully in our previous studies of membrane Adenylyl cyclases (ACs), (ii) the overall conformation of the AC catalytic domains stabilized by MANT-GTP is similar to that stabilized by ATP analogs, such as ATPαS, based on X-ray crystallographic studies[13,21]
designed ankyrin repeat protein (DARPin) are established as powerful agents for stabilization of macromolecules for structural and biochemical studies[20,28]

Summary

Introduction

6, Ana-Nicoleta Bondar[5,7], Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. 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 (AC5c1/AC2c2) in complex with Gαs and forskolin[12–14]. These studies provided the structural basis for the two metal-ioncatalysis 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]. The cryoEM structure of AC91250–Gαs bound to MANT-GTP and forskolin, combined with biochemical studies, confirmed that AC9 can be activated by forskolin binding to its canonical allosteric site in the presence of Gαs[1,17], further suggesting that AC9 is not completely insensitive to forskolin

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Results

Conclusion