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.
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We thank K. Hess and N. Stephanou for technical assistance and R. Abramowitz and X. Yang for support at National Synchrotron Light Source beamline X4A. C.S. acknowledges support as Berger Fellow of the Damon-Runyon Cancer Research Foundation, and H.W. is a Pew Scholar of Biomedical Sciences and a Rita Allen Scholar. This work was supported by funds from the US National Institutes of Health (L.R.L. and J.B.), Hirschl Weill-Caulier Trust (L.R.L.) and the Ellison Medical Foundation (J.B.).
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Kinetics of purified cyanobacterial sAC (CyaC). (PDF 41 kb)
Model of ATP–calcium bound to the sAC active site. (PDF 339 kb)
Bicarbonate activation of sAC at various pH values. (PDF 97 kb)
Active site of the sAC–Rp-ATPαS complex. (PDF 225 kb)
Stereospecific inhibition of sAC by ATPαS. (PDF 25 kb)
Effect of pH changes and anions on sAC activity and on sAC crystals. (PDF 12 kb)
Simulated transition between the open and closed form of sAC–α,β-Me-ATP. (MPG 1054 kb)
Model for catalysis by Class III nucleotidyl cyclases. (MPG 2926 kb)
Estimation of the off-rate for the sAC–bicarbonate complex. (PDF 19 kb)
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Steegborn, C., Litvin, T., Levin, L. et al. Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment. Nat Struct Mol Biol 12, 32–37 (2005). https://doi.org/10.1038/nsmb880
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