Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

Abstract

In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.The ubiquitous second messenger cAMP regulates a large variety of essential physiological processes such as gene expression, chromosome segregation and cellular metabolism. In mammalian cells, cAMP is synthesized by a family of nine transmembrane adenylyl cyclases (tmACs) and one sAC 1 . Unlike tmACs, which localize to the cellular membrane and respond to extracellular stimuli via heterotrimeric G proteins 1 , sAC is found in various intracellular compartments such as the mitochondria and the nucleus 2 . Its localization near intracellular cAMP targets is the impetus for current models of second messenger signal transduction, in which cAMP functions as a locally acting signaling molecule 2-4 .sAC is insensitive to the tmAC regulators calmodulin and heterotrimeric G proteins as well as the nonphysiological activator forskolin; instead, sAC senses physiological levels of bicarbonate 5 . Aside from its role as a universal physiological buffer maintaining cellular and extracellular pH, bicarbonate functions as a signaling molecule 3 , regulating many biological processes in mammals such as fertility 6 , acid-base homeostasis, breathing rate, metabolism and fluid transport (reviewed in ref. 7). As the only known signaling enzyme sensitive to physiological fluctuations of bicarbonate 5 , sAC probably mediates each of these processes. Bicarbonate activation of sAC is essential for sperm motility 8 as well as for pH-dependent COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. acid secretion in the epididymis and possibly the kidney 9 . In addition to its bicarbonate sensitivity, sAC is synergistically activated by calcium 10 , and this potentiation seems to be important for sperm maturation 11 . NIH Public AccessPrevious work has revealed the overall structure of tmAC enzymes and suggested a twometal ion mechanism for catalysis 12,13 . Despite their different regulation, mammalian sAC and tmACs are grouped into the nucleotidyl cyclase class III based on sequ...

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

“…The sAC-cat/ApCpp complex comprises only one metal ion, in position B, which supports substrate binding (3,23). Coordination number and distances identify it as a Ca 2+ , consistent with results on the cyanobacterial homolog CyaC and biochemical data showing that Ca 2+ stimulates sAC activity by increasing substrate affinity (8,13). In tmACs, a Ser (Ser1028 in rat tmACII-C 2 ) potentially contacts the ATP ribose ring oxygen (24,33).…”

“…1C), most of the seven conserved catalytic residues are arranged similar to other class III AC structures (23). However, one of the two conserved Asp residues responsible for coordinating magnesium, Asp99, is shifted from the catalytic position (13,24,33) in apo sAC ( Fig. 1C; CyaC Asp1061 is shown as a reference).…”

“…In a homodimeric, HCO 3 − -regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO 3 − appeared to facilitate an active site closure required for catalysis (13), but the HCO 3 − binding site and its mechanism of activation remained unknown.…”

“…; Ca 2+ supports substrate binding, and HCO 3 − increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3,13,29). In a homodimeric, HCO 3 − -regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO 3 − appeared to facilitate an active site closure required for catalysis (13), but the HCO 3 − binding site and its mechanism of activation remained unknown.…”

“…In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5-7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO 3 − ) (3,(8)(9)(10). HCO 3 − regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1,(11)(12)(13). Via modulation of sAC, and sAClike cyclase activities, HCO 3 − serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO 3 − , CO 2 , and pH (3,14).…”