Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

Abstract

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3−); it thereby serves as a cellular sensor for HCO3−, carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3− binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3− activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3− sensing and a basis for targeting this system with drugs.

Introducing a novel mechanism to control heart rate in the ancestral pacific hagfish

“…Indeed, tmAC may be nearly fully activated in normoxia given that forskolin, which stimulates cAMP and bypasses tmAC, failed to increase heart rate in either normoxia or beyond routine rates in anoxia. Importantly, forskolin does not fit into the pseudosymmetrical site at the sAC pseudodimer interface, which in tmACs results in stimulation of cAMP, and as a result sAC is insensitive to forskolin (Chen et al, 2000;Kleinboelting et al, 2014). The explanation for the inhibition of heart rate by the highest pharmacological dose of forskolin is unknown, but it resembles biphasic effects of forskolin reported in other systems (Szabo et al, 1990;Tian, et al, 2009).…”

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

“…3) is very similar to that of sGCa cat :sGCb cat (Allerston et al, 2013) and, to a lesser extent, sAC-C1-C2 (Kleinboelting et al, 2014a). If only the sequences between b1 and a4 (b19 and a49, respectively) are considered, more or less close superpositions are possible (Fig.…”

“…In human sAC, the first 470 amino acids comprise a short N-terminal tail as well as C1 and C2 connected by a linker of about 70 residues (Kleinboelting et al, 2014a). The C-terminal domain of variable length (human sAC ;1140 amino acids) contains a putative autoinhibitory domain, a P-site, and a protein/protein interaction domain.…”

“…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.…”