[go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Structural & Molecular Biology volume 12, pages 32–37 (2005)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Open conformation of sAC in complex with α,β-Me-ATP and calcium.
Figure 2: Bicarbonate induces active site closure.
Figure 3: Conformational states and comparison of AC enzymes.
Figure 4: Model for catalysis by class III nucleotidyl cyclases.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Hanoune, J. & Defer, N. Regulation and role of adenylyl cyclase isoforms. Annu. Rev. Pharmacol. Toxicol. 41, 145–174 (2001).

    Article  CAS  Google Scholar 

  2. Zippin, J.H. et al. Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains. FASEB J. 17, 82–84 (2003).

    Article  CAS  Google Scholar 

  3. Zippin, J.H. et al. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain. J. Cell Biol. 164, 527–534 (2004).

    Article  CAS  Google Scholar 

  4. Bundey, R.A. & Insel, P.A. Discrete intracellular signaling domains of soluble adenylyl cyclase: camps of cAMP? Sci. STKE 2004, PE19 (2004).

  5. Chen, Y. et al. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289, 625–628 (2000).

    Article  CAS  Google Scholar 

  6. Visconti, P.E. et al. Novel signaling pathways involved in sperm acquisition of fertilizing capacity. J. Reprod. Immunol. 53, 133–150 (2002).

    Article  CAS  Google Scholar 

  7. Zippin, J.H., Levin, L.R. & Buck, J. CO(2)/HCO(3)(−)-responsive soluble adenylyl cyclase as a putative metabolic sensor. Trends Endocrinol. Metab. 12, 366–370 (2001).

    Article  CAS  Google Scholar 

  8. Esposito, G. et al. Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc. Natl. Acad. Sci. USA 101, 2993–2998 (2004).

    Article  CAS  Google Scholar 

  9. Pastor-Soler, N. et al. Bicarbonate regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J. Biol. Chem. 278, 49523–49529 (2003).

    Article  CAS  Google Scholar 

  10. Litvin, T.N., Kamenetsky, M., Zarifyan, A., Buck, J. & Levin, L.R. Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate. J. Biol. Chem. 278, 15922–15926 (2003).

    Article  CAS  Google Scholar 

  11. Jaiswal, B.S. & Conti, M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa. Proc. Natl. Acad. Sci. USA 100, 10676–10681 (2003).

    Article  CAS  Google Scholar 

  12. Tesmer, J.J., Sunahara, R.K., Gilman, A.G. & Sprang, S.R. Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gαs.GTPγS. Science 278, 1907–1916 (1997).

    Article  CAS  Google Scholar 

  13. Tesmer, J.J. & Sprang, S.R. The structure, catalytic mechanism and regulation of adenylyl cyclase. Curr. Opin. Struct. Biol. 8, 713–719 (1998).

    Article  CAS  Google Scholar 

  14. Linder, J.U. & Schultz, J.E. The class III adenylyl cyclases: multi-purpose signalling modules. Cell Signal. 15, 1081–1089 (2003).

    Article  CAS  Google Scholar 

  15. Buck, J., Sinclair, M.L., Schapal, L., Cann, M.J. & Levin, L.R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA 96, 79–84 (1999).

    Article  CAS  Google Scholar 

  16. Tesmer, J.J. et al. Two-metal-Ion catalysis in adenylyl cyclase. Science 285, 756–760 (1999).

    Article  CAS  Google Scholar 

  17. Eckstein, F., Romaniuk, P.J., Heideman, W. & Storm, D.R. Stereochemistry of the mammalian adenylate cyclase reaction. J. Biol. Chem. 256, 9118–9120 (1981).

    CAS  PubMed  Google Scholar 

  18. Fox, B.A. et al. Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by X-ray crystallography. J. Biol. Chem. 274, 4917–4923 (1999).

    Article  CAS  Google Scholar 

  19. Pidcock, E. & Moore, G.R. Structural characteristics of protein binding sites for calcium and lanthanide ions. J. Biol. Inorg. Chem. 6, 479–489 (2001).

    Article  CAS  Google Scholar 

  20. Katz, A.K., Glusker, J.P., Beebe, S.A. & Bock, C.W. Calcium ion coordination: A comparison with that of beryllium, magnesium, and zinc. J. Am. Chem. Soc. 118, 5752–5763 (1996).

    Article  CAS  Google Scholar 

  21. Mohan, M.S. & Rechnitz, G.A. Ion-electrode study of the calcium-adenosine triphosphate system. J. Am. Chem. Soc. 94, 1714–1716 (1972).

    Article  CAS  Google Scholar 

  22. Yan, S.-Z., Huang, Z.-H., Shaw, R.S. & Tang, W.-J. The conserved asparagine and arginine are essential for catalysis of mammalian adenylyl cyclase. J. Biol. Chem. 272, 12342–12349 (1997).

    Article  CAS  Google Scholar 

  23. Garbers, D.L. & Johnson, R.A. Metal and metal-ATP interactions with brain and cardiac adenylate cyclase. J. Biol. Chem. 250, 8449–8456 (1975).

    CAS  PubMed  Google Scholar 

  24. Cann, M.J., Hammer, A., Zhou, J. & Kanacher, T. A defined subset of adenylyl cyclases is regulated by bicarbonate ion. J. Biol. Chem. 278, 35033–35038 (2003).

    Article  CAS  Google Scholar 

  25. Kasahara, M., Yashiro, K., Sakamoto, T. & Ohmori, M. The Spirulina platensis adenylate cyclase gene, cyaC, encodes a novel signal transduction protein. Plant Cell Physiol. 38, 828–836 (1997).

    Article  CAS  Google Scholar 

  26. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  27. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  28. Jogl, G., Tao, X., Xu, Y. & Tong, L. COMO: a program for combined molecular replacement. Acta Crystallogr. D 57, 1127–1134 (2001).

    Article  CAS  Google Scholar 

  29. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  30. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  31. Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. Sect. D 54, 905–921 (1998).

    Article  Google Scholar 

  32. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein strucrures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  33. Merrit, E.A. & Murphy, M.E.P. RASTER3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr. Sect. D 50, 869–873 (1994).

    Article  Google Scholar 

  34. Evans, S.V. SETOR: hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134–138 (1993).

    Article  CAS  Google Scholar 

  35. Barton, G.J. ALSCRIPT a tool to format multiple sequence alignments. Prot. Engineering 6, 37–40 (1993).

    Article  CAS  Google Scholar 

Download references

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

  1. Tatiana N Litvin

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

Authors and Affiliations

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

    Clemens Steegborn & Hao Wu

  2. Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, 10021, New York, USA

    Tatiana N Litvin, Lonny R Levin & Jochen Buck

Authors
  1. Clemens Steegborn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tatiana N Litvin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lonny R Levin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jochen Buck
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hao Wu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Kinetics of purified cyanobacterial sAC (CyaC). (PDF 41 kb)

Supplementary Fig. 2

Model of ATP–calcium bound to the sAC active site. (PDF 339 kb)

Supplementary Fig. 3

Bicarbonate activation of sAC at various pH values. (PDF 97 kb)

Supplementary Fig. 4

Active site of the sAC–Rp-ATPαS complex. (PDF 225 kb)

Supplementary Fig. 5

Stereospecific inhibition of sAC by ATPαS. (PDF 25 kb)

Supplementary Table 1

Effect of pH changes and anions on sAC activity and on sAC crystals. (PDF 12 kb)

Supplementary Video 1

Simulated transition between the open and closed form of sAC–α,β-Me-ATP. (MPG 1054 kb)

Supplementary Video 2

Model for catalysis by Class III nucleotidyl cyclases. (MPG 2926 kb)

Supplementary Methods

Estimation of the off-rate for the sAC–bicarbonate complex. (PDF 19 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb880

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing