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

Nature Structural & Molecular Biology volume 12, pages 3237 (2005) | Download Citation

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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.

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Acknowledgements

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.).

Author information

Author notes

    • Tatiana N Litvin

    Present address: Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, USA.

Affiliations

  1. Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.

    • Clemens Steegborn
    •  & Hao Wu
  2. Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.

    • Tatiana N Litvin
    • , Lonny R Levin
    •  & Jochen Buck

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hao Wu.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Kinetics of purified cyanobacterial sAC (CyaC).

  2. 2.

    Supplementary Fig. 2

    Model of ATP–calcium bound to the sAC active site.

  3. 3.

    Supplementary Fig. 3

    Bicarbonate activation of sAC at various pH values.

  4. 4.

    Supplementary Fig. 4

    Active site of the sAC–Rp-ATPαS complex.

  5. 5.

    Supplementary Fig. 5

    Stereospecific inhibition of sAC by ATPαS.

  6. 6.

    Supplementary Table 1

    Effect of pH changes and anions on sAC activity and on sAC crystals.

  7. 7.

    Supplementary Methods

    Estimation of the off-rate for the sAC–bicarbonate complex.

Videos

  1. 1.

    Supplementary Video 1

    Simulated transition between the open and closed form of sAC–α,β-Me-ATP.

  2. 2.

    Supplementary Video 2

    Model for catalysis by Class III nucleotidyl cyclases.

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DOI

https://doi.org/10.1038/nsmb880

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