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:

Crystal structure of recombinant bovine neurocalcin

Abstract

The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 Å resolution. The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca 2+ -binding loop. The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ. Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase. A possible site for cyclase activity based on the three-dimensional structure is discussed.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Amino acid sequence alignment of bovNca (bovine neurocalcin) with recoverin-like proteins.
Figure 2: Structural depictions of neurocalcin.
Figure 3: Representation of the four EF-hands of neurocalcin (EF1, EF2, EF3, EF4).
Figure 4
Figure 5: Stereo view of 2|Fo - Fc| electron density map contoured at 1.5σ for residues 72-78 of the EF2-hand.
Figure 6: EF-hand structures.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Kretsinger, R.H. Hypothesis: Calcium modulated proteins contain EF-hands. In Calcium transport in contraction and secretion (ed. Carafoli, E.) 469–480 (North-Holland Publishing, Amsterdam; 1985).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Polans, A., Baehr, W. & Palczewski, K. Turned on by Ca2+! The physiology and pathology of Ca2+-binding proteins in the retina. Trends Neurosci. 19, 547–554 (1996).

    Article  CAS  Google Scholar 

  4. Dizhoor, A.M. et al. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science 251, 915–918 (1991).

    Article  CAS  Google Scholar 

  5. Gray-Keller, M.P., Polans, A.S., Palczewski, K. & Detwiler, P.B. The effect of recoverin-like calcium binding proteins on the photoresponse of retinal rods. Neuron 10, 523–531 (1993).

    Article  CAS  Google Scholar 

  6. Kawamura, S. & Murakami, M. Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods. Nature 349, 420–423 (1991).

    Article  CAS  Google Scholar 

  7. Polans, A., Baehr, W. & Palczewski, K. Turned on by Ca2+! The physiology and pathology of Ca2+-binding proteins in the retina. 19, 547–554 (1996).

  8. Kawamura, S., Takamatsu, K. & Kitamura, K. Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod receptors. Biochem. Biophys. Res. Commun. 186, 411–417 (1992).

    Article  CAS  Google Scholar 

  9. Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H. & Miki, N. Visinin: a novel calcium binding protein expressed in retinal cone cells. Neuron 2, 469–478 (1990).

    Article  Google Scholar 

  10. Pongs, O. et al. Frequenin: A novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system. Neuron 11, 15–28 (1993).

    Article  CAS  Google Scholar 

  11. Kobayashi, M., Takamatsu, K., Saitoh, S., Mirra, M. & Noguchi, T. Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus. Biochem. Biophys. Res. Commun. 189, 511–517 (1992).

    Article  CAS  Google Scholar 

  12. Lenz, S.E., Henschel, Y., Zopf, D., Voss, B. & Gundelfinger, E.D. VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the chicken brain. Brain Res. Mol. Brain Res. 15, 133–140 (1992).

    Article  CAS  Google Scholar 

  13. Terasawa, M., Nakano, A., Kobayashi, R. & Hidaka, H. Neurocalcin: a novel calcium binding protein from bovine brain. J. Biol. Chem. 267, 19596–19599 (1992).

    CAS  PubMed  Google Scholar 

  14. Okazaki, K. et al. Full sequence of neurocalcin, a novel calcium binding protein abundant in the central nervous system. Biochem. Biophys. Res. Commun. 185, 147–153 (1992).

    Article  CAS  Google Scholar 

  15. Dyer, J.R., Sossin, W.S. & Klein, M. Cloning and characterization of aplycalcin and aplysia neurocalcin, two new members of the calmodulin superfamily of small calcium-binding proteins. J. Neurochem. 67, 932–942 (1996).

    Article  CAS  Google Scholar 

  16. Dizhoor, A.M. et al. Cloning sequencing and expression of 24-kDa Ca2+-binding protein activating photoreceptor guanlyl cyclase. J. Biol. Chem. 27, 25200–25206 (1995).

    Article  Google Scholar 

  17. Gorczyca, W.A., Gray-Keller, M.P., Detwiler, P.B. & Palczewski, K. Purification and physiological evaluation of guanylate cyclase activating protein from retinal rods. Proc. Natl. Acad. Sci. USA 91, 4014–4018 (1994).

    Article  CAS  Google Scholar 

  18. Nakano, A. et al. Distinct regional localization of neurocalcin, a Ca2+-binding protein, in the bovine adrenal gland. J. Endocrinol. 138, 283–290 (1993).

    Article  CAS  Google Scholar 

  19. Ilino, S., Kobayashi, S., Okazaki, K. & Hidaka, H. Immunohistochemical localization of neurocalcin in the rat inner ear. Brain Res. 680, 128–134 (1995).

    Article  Google Scholar 

  20. Ilino, S., Kobayashi, S., Okazaki, K. & Hidaka, H. Neurocalcin-immunoreactive receptor cells in the rat olfactory epithelium and vomeronasal organ. Neurosci. Lett. 191, 91–94 (1995).

    Article  Google Scholar 

  21. Ladant, D. Calcium and membrane binding properties of bovine neurocalcin d expressed in Escherichia coli. J. Biol. Chem. 270, 3179–3185 (1995).

    CAS  PubMed  Google Scholar 

  22. Teng, D.H.-F., Chen, C.-K. & Hurley, J.B. A highly conserved homologue of bovine neurocalcin in Drosophila melanogaster is a Ca2+-binding protein expressed in neuronal tissues. J. Biol. Chem. 269, 31900–31907 (1994).

    CAS  PubMed  Google Scholar 

  23. 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  CAS  Google Scholar 

  24. Ames, J.B. et al. Molecular mechanics of calcium-myristoyl switches. Nature 389, 198–202 (1997).

    Article  CAS  Google Scholar 

  25. Furobert, E., Chen, C.-K., Hurley, J.B. & Teng, D.H.-F. Drosophila neurocalcin, a fatty acylated Ca2+-binding protein that associates with membranes and inhibits in vitro phosphorylation of bovine rhodopsin. J. Biol. Chem. 271, 10256–10262 (1996).

    Article  Google Scholar 

  26. Babu, Y.S., Bugg, C.E. & Cook, W.J. Structure of calmodulin refined at 2.2 Å resolution. J. Mol. Biol. 204, 191–204 (1988).

    Article  CAS  Google Scholar 

  27. Blanchard, H. et al. Structure of Ca2+-binding domain reveals a novel EF-hand and Ca2+-induced conformational changes. Nature Struct. Biol. 4, 532–538 (1997).

    Article  CAS  Google Scholar 

  28. Jones, S. & Thornton, J.M. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93, 13–20 (1996).

    Article  CAS  Google Scholar 

  29. Resh, M.D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell 76, 411–413 (1994).

    Article  CAS  Google Scholar 

  30. McLauglin, S. & Aderem, A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20, 272–276 (1995).

    Article  Google Scholar 

  31. Flaherty, K.M., Zozulya, S., Stryer, L. & McKay, D.B. Three-dimensional structure of recoverin, a calcium sensor in vision. Cell. 75, 709–716 (1993).

    Article  CAS  Google Scholar 

  32. Herzberg, O. & James, M.N.G. Refined crystal structure of troponin C from turkey muscle at 2.0 Å resolution. J. Mol. Biol. 203, 761–77 (1988).

    Article  CAS  Google Scholar 

  33. Vijay-Kumar, S. & Cook, W.L. Structure of sacoplasmic calcium-binding protein from Nersi Diversicolor refined at 2.0 Å resolution. J. Mol. Biol. 224, 413–426 (1992).

    Article  CAS  Google Scholar 

  34. Duda, T., Goraczniak, R.M. & Sharma, R.K. Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase. Biochemistry 35, 6263–6266 (1996).

    Article  CAS  Google Scholar 

  35. Kumar, V.D., Hidaka, H., Okazaki, K. & Vijay-Kumar, S. Crystallization and preliminary X-ray crystallographic studies of recombinant bovine neurocalcin d. Proteins 25, 261–264 (1996).

    Article  CAS  Google Scholar 

  36. Thaller, C. et al. Repeated seeding technique for growing large single crystals of proteins. J. Mol. Biol. 147, 465–469 (1981).

    Article  CAS  Google Scholar 

  37. Higashi, T. PROCESS: A program for indexing and processing RAXIS IIC imaging plate data. (Rigaku Corporation, Japan; 1990).

  38. Wonacott, A.J. In The rotation method in crystallography (eds Arndt, U.W. & Wonacott, A.J.) 75–103 (Amsterdam, North-Holland; 1971).

    Google Scholar 

  39. Sheldrick. G.M. & Schneider, T.R. SHELXL: high resolution refinement. Meth. Enz. 277, 319–343 (1997).

    Article  CAS  Google Scholar 

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

  41. Jones, T.A. Interactive computer graphics: FRODO. Meth. Enz. 115, 157–171 (1985).

    Article  CAS  Google Scholar 

  42. Cowtan, K.D. & Main, P. Improvement of macromolecular electron density maps by the simultaneous application of real and reciprocal space constraints. Acta Crystallogr. D 49, 148–157 (1993).

    Article  CAS  Google Scholar 

  43. Read, R.J. Improved fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986).

    Article  Google Scholar 

  44. Brunger, A.T. XPLOR version 3.1, A System For X-Ray Crystallography And NMR (Yale University Press, New Haven, Connecticut; 1992).

    Google Scholar 

  45. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK-a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–191(1993).

    Article  CAS  Google Scholar 

  46. Kraulis, P. MOLSCRIPT — a program to produce detailed and schematic plots for protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  47. Merrit, E.A. & Murphy, M.E. Raster3D Version 2.0. A program for photo-realistic molecular graphics. Acta Crystallogr. D 50, 869–873 (1994).

    Google Scholar 

Download references

Acknowledgements

We thank I. Weber for comments on the manuscript. This work was funded in part by Markey's foundation (S.V.-K.) and Kimmel Cancer Center (V.D.K).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Vinod D. Kumar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vijay-Kumar, S., Kumar, V. Crystal structure of recombinant bovine neurocalcin. Nat Struct Mol Biol 6, 80–88 (1999). https://doi.org/10.1038/4956

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/4956

This article is cited by

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