Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

Abstract

Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…At present, the PDB provides 28 sAC crystal structures (Kleinboelting et al, 2014a(Kleinboelting et al, ,b, 2016Saalau-Bethell et al, 2014;RamosEspiritu et al, 2016), including complexes with ATP, a,b-methylene adenosine-59-triphosphate, cAMP, pyrophosphate, bicarbonate, biselenite, bisulfite, bithionol, and various inhibitors. Comparing structures of 5C1: 2C2, of sAC-C1-C2 and of an inactive human sGCa cat : sGCb cat heterodimer (Allerston et al, 2013;Seeger et al, 2014) indicates high structural homology not only of the individual catalytic domains, but also of the whole complexes, and points to a common, well conserved mechanism of cNMP generation facilitated by two metal ions.…”

Detection of secondary binding sites in proteins using fragment screening

“…It was subsequently shown that L-serine binding to this allosteric site activates PKM2. Finally, the allosteric bicarbonate binding site of human soluble adenylate cyclase (SolAC) was also characterized using X-ray crystallography (20,21), and a subsequent fragment screen against this target identified inhibitors that occupy this site.…”

Role of soluble adenylyl cyclase in mitochondria

“…Taken together, these findings suggest that sAC can be considered as a putative metabolic sensor. Recently the crystal structure of human sAC apo state was described [8, 9], including the mechanism of catalysis and the binding site of the HCO 3 − activation and regulators. The structure shows how HCO 3 − binds and activates sAC and how sAC can be inhibited by a drug [8, 9].…”