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Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein

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Abstract

Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein ( CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three “EF-hand” Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 Å, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.

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References

  1. J. G. Morin, in Coelenterate Biology: Reviews and New Perspectives ed. L. Muscatine and H. M. Lenhoff, Academic Press, New York, 1974, pp. 397–438.

  2. C. M. Thomson, P. J. Herring, A. K. Campbell, The widespread occurrence and tissue distribution of the imidazolopyrazine luciferins, J. Biolum. Chemilum., 1997, 12, 87–91.

    Article  CAS  Google Scholar 

  3. Y. Ohmiya, T. Hirano, Shining the light: The mechanism of the bioluminescence reaction of calcium-binding photoproteins, Chem. Biol., 1996, 3, 337–347.

    Article  CAS  PubMed  Google Scholar 

  4. O. Shimomura, F. H. Johnson, Y. Saiga, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea, J. Cell. Comp. Physiol., 1962, 59, 223–239.

    Article  CAS  PubMed  Google Scholar 

  5. O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing Co., Singapore, 2006.

    Book  Google Scholar 

  6. J. G. Morin, J. W. Hastings, Biochemistry of the bioluminescence of colonial hydroids and other coelenterates, J. Cell. Physiol., 1971, 77, 305–312.

    Article  CAS  PubMed  Google Scholar 

  7. A. K. Campbell, Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata, Biochem. J., 1974, 143, 411–418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. E. S. Vysotski, V. S. Bondar, V. N. Letunov, Extraction and purification of obelin, the Ca2+-dependent photoprotein from the hydroid Obelia longissima, Biochemistry (Moscow), 1989, 54, 965–973.

    Google Scholar 

  9. N. D. Moncrief, R. H. Kretsinger, M. Goodman, Evolution of EF-hand calcium-modulated proteins. I. Relationships, based on amino acid sequences, J. Mol. Evol., 1990, 30, 522–562.

    Article  CAS  PubMed  Google Scholar 

  10. J. C. Matthews, K. Hori, M. J. Cormier, Purification and properties of Renilla reniformis luciferase, Biochemistry, 1977, 16, 85–91.

    Article  CAS  PubMed  Google Scholar 

  11. S. Inouye, K. Watanabe, H. Nakamura, O. Shimomura, Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase, FEBS Lett., 2000, 481, 19–25.

    Article  CAS  PubMed  Google Scholar 

  12. O. Shimomura, P. R. Flood, S. Inouye, B. Bryan, A. Shimomura, Isolation and properties of the luciferase stored in the ovary of the scyphozoan medusa Periphylla periphylla, Biol. Bull., 2001, 201, 339–347.

    Article  CAS  PubMed  Google Scholar 

  13. S. V. Markova, S. Golz, L. A. Frank, B. Kalthof, E. S. Vysotski, Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa, A novel secreted bioluminescent reporter enzyme, J. Biol. Chem., 2004, 279, 3212–3217.

    Article  CAS  PubMed  Google Scholar 

  14. H. Charbonneau, K. A. Walsh, R. O. McCann, F. G. Prendergast, M. J. Cormier, T. C. Vanaman, Amino acid sequence of the calcium-dependent photoprotein aequorin, Biochemistry, 1985, 24, 6762–6771.

    Article  CAS  PubMed  Google Scholar 

  15. E. S. Vysotski, J. Lee, Ca2+-regulated photoproteins: Structural insight into the bioluminescence mechanism, Acc. Chem. Res., 2004, 37, 405–415.

    Article  CAS  PubMed  Google Scholar 

  16. F. I. Tsuji, Y. Ohmiya, T. F. Fagan, H. Toh, S. Inouye, Molecular evolution of the Ca2+-binding photoproteins of the Hydrozoa, Photochem. Photobiol., 1995, 62, 657–661.

    Article  CAS  PubMed  Google Scholar 

  17. S. V. Markova, E. S. Vysotski, J. R. Blinks, L. P. Burakova, B. C. Wang, J. Lee, Obelin from the bioluminescent marine hydroid Obelia geniculata: Cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins, Biochemistry, 2002, 41, 2227–2236.

    Article  CAS  PubMed  Google Scholar 

  18. W. W. Lorenz, R. O. McCann, M. Longiaru, M. J. Cormier, Isolation and expression of a cDNA encoding Renilla reniformis luciferase, Proc. Natl. Acad. Sci. USA, 1991, 88, 4438–4442.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. J. W. Hastings, J. C. Dunlap, Cell-free components in dinoflagellate bioluminescence. The particulate activity: scintillons; the soluble components: luciferase; luciferin, and luciferin-binding protein, Methods Enzymol., 1986, 133, 307–327.

    Article  CAS  Google Scholar 

  20. H. Charbonneau, M. J. Cormier, Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein, J. Biol. Chem., 1979, 254, 769–780.

    Article  CAS  PubMed  Google Scholar 

  21. W. W. Ward, M. J. Cormier, An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein, J. Biol. Chem., 1979, 254, 781–788.

    Article  CAS  PubMed  Google Scholar 

  22. S. Kumar, M. Harrylock, K. A. Walsh, M. J. Cormier, H. Charbonneau, Amino acid sequence of the Ca2+-triggered luciferin binding protein of Renilla reniformis, FEBS Lett., 1990, 268, 287–290.

    Article  CAS  PubMed  Google Scholar 

  23. K. Hori, J. E. Wampler, J. C. Matthews, M. J. Cormier, Identification of the product excited states during the chemiluminescent and bioluminescent oxidation of Renilla (sea pansy) luciferin and certain of its analogs, Biochemistry, 1973, 12, 4463–4468.

    Article  CAS  PubMed  Google Scholar 

  24. W. W. Ward, M. J. Cormier, In vivo energy transfer in Renilla bioluminescence, J. Phys. Chem., 1976, 80, 2289–2291.

    Article  CAS  Google Scholar 

  25. B. A. Illarionov, V. S. Bondar, V. A. Illarionova, E. S. Vysotski, Sequence of the cDNA encoding the Ca2+-activated photoprotein obelin from the hydroid polyp Obelia longissima, Gene, 1995, 153, 273–274.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. J. F. Head, S. Inouye, K. Teranishi, O. Shimomura, The crystal structure of the photoprotein aequorin at 2.3 Å resolution, Nature, 2000, 405, 372–376.

    Article  CAS  PubMed  Google Scholar 

  28. Z. J. Liu, E. S. Vysotski, C. J. Chen, J. Rose, J. Lee, B. C. Wang, Structure of the Ca2+-regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure, Protein Sci., 2000, 9, 2085–2093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. L. Deng, S. V. Markova, E. S. Vysotski, Z. J. Liu, J. Lee, J. Rose, B. C. Wang, Crystal structure of a Ca2+-discharged photoprotein: Implications for mechanisms of the calcium trigger and bioluminescence, J. Biol. Chem., 2004, 279, 33647–33652.

    Article  CAS  PubMed  Google Scholar 

  30. Z. J. Liu, G. A. Stepanyuk, E. S. Vysotski, J. Lee, S. V. Markova, N. P. Malikova, B. C. Wang, Crystal structure of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary excited state, Proc. Natl. Acad. Sci. USA, 2006, 103, 2570–2575.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. L. Deng, E. S. Vysotski, S. V. Markova, Z. J. Liu, J. Lee, J. Rose, B. C. Wang, All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin, Protein Sci., 2005, 14, 663–675.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. M. S. Titushin, S. V. Markova, L. A. Frank, N. P. Malikova, G. A. Stepanyuk, J. Lee, E. S. Vysotski, Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase, Photochem. Photobiol. Sci., 2008, 7, 189–196.

    Article  CAS  PubMed  Google Scholar 

  33. F. W. Studier, Protein production by auto-induction in high-density shaking cultures, Protein Expression Purif., 2005, 41, 207–234.

    Article  CAS  Google Scholar 

  34. E. S. Vysotski, Z. J. Liu, J. Rose, B. C. Wang, J. Lee, Preparation and X-ray crystallographic analysis of recombinant obelin crystals diffracting to beyond 1.1 Å, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2001, 57, 1919–1921.

    Article  CAS  Google Scholar 

  35. Z. J. Liu, W. Tempel, J. D. Ng, D. Lin, A. K. Shah, L. Chen, P. S. Horanyi, J. E. Habel, I. A. Kataeva, H. Xu et al., The high-throughput protein-to-structure pipeline at SECSG, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2005, 61, 679–684.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  37. T. C. Terwilliger, SOLVE and RESOLVE: automated structure solution and density modification, Methods Enzymol., 2003, 374, 22–37.

    Article  CAS  PubMed  Google Scholar 

  38. T. C. Terwilliger, J. Berendzen, Automated MAD and MIR structure solution, Acta Crystallogr., Sect. D: Biol. Crystallogr., 1999, 55, 849–861.

    Article  CAS  Google Scholar 

  39. Z. J. Liu, D. Lin, W. Tempel, J. L. Praissman, J. P. Rose, B. C. Wang, Parameter-space screening: a powerful tool for high-throughput crystal structure determination, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2005, 61, 520–527.

    Article  CAS  Google Scholar 

  40. A. Perrakis, M. Harkiolaki, K. S. Wilson, V. S. Lamzin, ARP/wARP and molecular replacement, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2001, 57, 1445–1450.

    Article  CAS  Google Scholar 

  41. I. W. Davis, L. W. Murray, J. S. Richardson, D. C. Richardson, MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes, Nucleic Acids Res., 2004, 32 Web Server issue, W615–619.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. W. B. Arendall 3rd, W. Tempel, J. S. Richardson, W. Zhou, S. Wang, I. W. Davis, Z. J. Liu, J. P. Rose, W. M. Carson, M. Luo et al., A test of enhancing model accuracy in high-throughput crystallography, J. Struct. Funct. Genomics, 2005, 6, 1–11.

    Article  CAS  PubMed  Google Scholar 

  43. D. E. McRee, XtalView/Xfit - A versatile program for manipulating atomic coordinates and electron density, J. Struct. Biol., 1999, 125, 56–65.

    Article  Google Scholar 

  44. M. D. Winn, G. N. Murshudov, M. Z. Papiz, Macromolecular TLS refinement in REFMAC at moderate resolutions, Methods Enzymol., 2003, 374, 300–321.

    Article  CAS  PubMed  Google Scholar 

  45. E. Potterton, P. Briggs, M. Turkenburg, E. Dodson, A graphical user interface to the CCP4 program suite, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2003, 59, 1131–1137.

    Article  Google Scholar 

  46. H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, The Protein Data Bank, Nucleic Acids Res., 2000, 28, 235–242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. N. C. Strynadka and M. N. James, in Encyclopedia of Inorganic Chemistry, ed. R. B. King, John Wiley & Sons, New York, 1994, pp. 477–507.

  48. F. McCapra, Y. C. Chang, The chemiluminescence of a Cypridina luciferin analogue, Chem. Commun., 1967, 19, 1011–1012.

    Google Scholar 

  49. T. Goto, Chemistry of bioluminescence, Pure Appl. Chem., 1968, 17, 421–441.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    Galina A. Stepanyuk, John Lee, Eugene S. Vysotski & Bi-Cheng Wang

  2. Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences Siberian Branch, Krasnoyarsk, 660036, Russia

    Galina A. Stepanyuk, Svetlana S. Markova, Ludmila A. Frank & Eugene S. Vysotski

  3. National Lab of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing, 100101, China

    Zhi-Jie Liu

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  1. Galina A. Stepanyuk
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Correspondence to Zhi-Jie Liu.

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Stepanyuk, G.A., Liu, ZJ., Markova, S.S. et al. Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein. Photochem Photobiol Sci 7, 442–447 (2008). https://doi.org/10.1039/b716535h

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