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.

  • Letter
  • Published:

The crystal structure of the photoprotein aequorin at 2.3 Å resolution

Nature volume 405, pages 372–376 (2000)Cite this article

Abstract

Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea1. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix–loop–helix ‘EF-hand’ domains, of which three can bind calcium2. The molecule also contains coelenterazine as its chromophoric ligand3. When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light4. Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent5. Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described7; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity8, permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Ribbon representation showing the secondary structure elements in the protein.
Figure 2: Model of peroxidized coelenterazine with Tyr 184 and His 169.
Figure 3: Stereo view of peroxidized coelenterazine (at centre) with some of the surrounding residues.
Figure 4: Representation of peroxidized coelenterazine showing all distances to protein atoms within 3.6 Å and some other local interactions.

Similar content being viewed by others

References

  1. Shimomura, O., Johnson, F. H. & Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223– 240 (1962).

    Article  CAS  Google Scholar 

  2. Inouye, S. et al. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc. Natl Acad. Sci. 82, 3145 –3158 (1985).

    Article  ADS  Google Scholar 

  3. Shimomura, O. & Johnson, F. H. Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc. Natl Acad. Sci. USA 75, 2611–2615 ( 1978).

    Article  ADS  CAS  Google Scholar 

  4. Shimomura, O. & Johnson, F. H. Chemical nature of light emitter in bioluminescence of aequorin. Tetrahedron Lett. 31 , 2963–2966 (1973).

    Article  Google Scholar 

  5. Shimomura, O. & Johnson, F. H. Regeneration of the photoprotein aequorin. Nature 256, 236– 238 (1975).

    Article  ADS  CAS  Google Scholar 

  6. Prasher, D., McCann, R. O. & Cormier, M. J. Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem. Biophys. Res. Commun. 126, 1259–1263 ( 1985).

    Article  CAS  Google Scholar 

  7. Hannick, L. I., Prasher, D. C., Schultz, L. W., Deschamps, J. R. & Ward, K. B. Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria. Proteins Struct. Funct. Genet. 15, 103–107 (1993).

    Article  CAS  Google Scholar 

  8. Shimomura, O. & Inouye, S. The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin. Protein Expr. Purif. 16, 91–95 (1999).

    Article  CAS  Google Scholar 

  9. Tanaka, T., Ames, J. B., Harvey, T. S., Stryer, L. & Ikura, M. Sequestration of the membrane-targeting myristoyl group of recoverin in the calcium-free state. Nature 376, 444–447 ( 1995).

    Article  ADS  CAS  Google Scholar 

  10. Vijay-Kumar, S. & Cook, W. J. Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2. 0 A resolution. J. Mol. Biol. 224, 413– 426 (1992).

    Article  CAS  Google Scholar 

  11. Cook, W. J., Jeffrey, L. C., Cox, J. A. & Vijay-Kumar, S. Structure of a sarcoplasmic calcium-binding protein from amphioxus refined at 2. 4 Ångstroms resolution. J. Mol. Biol. 229 , 461–471 (1993).

    Article  CAS  Google Scholar 

  12. Musicki, B., Kishi, Y. & Shimomura, O. Structure of functional part of photoprotein aequorin. J. Chem. Soc. Chem. Commun. 126, 1256– 1268 (1986).

    Google Scholar 

  13. Ohmiya, Y. & Tsuji, F. I. Bioluminescence of the Ca2+-binding photoprotein, aequorin, after histidine modification. FEBS Lett. 320, 267–270 (1993).

    Article  CAS  Google Scholar 

  14. Ohmiya, Y., Ohashi, M. & Tsuji, F. I. Two excited states in aequorin bioluminescence induced by tryptophan modification. FEBS Lett. 301, 197–201 (1992).

    Article  CAS  Google Scholar 

  15. Tsuji, F. I., Inouye, S., Goto, T. & Sakaki, Y. Site-specific mutagenesis of the calcium-binding photoprotein aequorin. Proc. Natl Acad. Sci. 83, 8107–8111 ( 1986).

    Article  ADS  CAS  Google Scholar 

  16. Shimomura, O., Musicki, B. & Kishi, Y. Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration. Biochem. J. 251, 405–410 (1988).

    Article  CAS  Google Scholar 

  17. Shimomura, O., Musicki, B. & Kishi, Y. Semi-synthetic aequorins with improved sensitivity to Ca2+ ions. Biochem. J. 261, 913–920 (1989).

    Article  CAS  Google Scholar 

  18. Shimomura, O., Inouye, S., Musicki, B. & Kishi, Y. Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators. Biochem. J. 270, 309– 312 (1990).

    Article  CAS  Google Scholar 

  19. Shimomura, O. Luminescence of aequorin is triggered by the binding of two calcium ions. Biochem. Biophys. Res. Commun. 211, 359 –363 (1995).

    Article  CAS  Google Scholar 

  20. Shimomura, O. & Inouye, S. Titration of recombinant aequorin with calcium chloride. Biochem. Biophys. Res. Commun. 221, 77–81 (1996).

    Article  CAS  Google Scholar 

  21. Nomura, M., Inouye, S., Ohmiya, Y. & Tsuji, F. I. A C-terminal proline is required for bioluminescence of the Ca2+-binding photoprotein, aequorin. FEBS Lett. 295, 63–66 (1991).

    Article  CAS  Google Scholar 

  22. Inouye, S., Aoyama, S., Miyata, T., Tsuji, F. I. & Sakaki, Y. Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin. J. Biochem. (Tokyo) 105, 473–477 (1989).

    Article  CAS  Google Scholar 

  23. Otwinowski, Z. in Proceedings of the CCP4 Study Weekend: Data Collection and Processing. (eds Sawyers, L. Isaacs, N. & Bailey, S.) 56– 62 (SERC Daresbury Laboratory, Warrington, UK; 1993 ).

    Google Scholar 

  24. Terwilliger, T. C. & Berendzen, J. Automated structure solution for MIR and MAD. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  25. Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 ( 1994).

    Article  Google Scholar 

  26. 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).

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

    Article  CAS  Google Scholar 

  28. Kleywegt, G. J. & Jones, T. A. Databases in protein crystallography. Acta Crystallogr. D 54, 1119–1131 (1998).

    Article  CAS  Google Scholar 

  29. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18, 2714–2723 ( 1997).

    Article  CAS  Google Scholar 

  30. POVRAY. Persistence of Vision Raytracer version 3. 1. 〈http://www.povray.org〉

Download references

Acknowledgements

We thank B. Kaminer for initiating this collaborative effort; B. Seaton for involvement in early crystallization studies; H. Nakamura for structural information on imidazopyrazinone; and the Boston University Mass Spectrometry Facility. This work was supported in part by NSF grants to O.S. and to J.F.H.

Author information

Authors and Affiliations

  1. Department of Physiology, Structural Biology Group, Boston University School of Medicine, Boston, 02118, Massachussetts, USA

    James F. Head & Osamu Shimomura

  2. Yokohama Research Centre, Chisso Corporation, 5-1 Okawa, Kanazawa-ku, 236, Yokohama, Japan

    Satoshi Inouye

  3. Faculty of Bioresources, Mie University, Kamihama, 514, Tsu, Japan

    Katsunori Teranishi

  4. Marine Biologicals Laboratory, Woods Hole, 02543, Massachussetts, USA

    Osamu Shimomura

Authors
  1. James F. Head
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satoshi Inouye
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katsunori Teranishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Osamu Shimomura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James F. Head.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Head, J., Inouye, S., Teranishi, K. et al. The crystal structure of the photoprotein aequorin at 2.3 Å resolution . Nature 405, 372–376 (2000). https://doi.org/10.1038/35012659

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35012659

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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