Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Dec;8(12):2580–2588. doi: 10.1110/ps.8.12.2580

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

H Aitio 1, A Annila 1, S Heikkinen 1, E Thulin 1, T Drakenberg 1, I Kilpeläinen 1
PMCID: PMC2144237  PMID: 10631973

Abstract

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babu Y. S., Bugg C. E., Cook W. J. Structure of calmodulin refined at 2.2 A resolution. J Mol Biol. 1988 Nov 5;204(1):191–204. doi: 10.1016/0022-2836(88)90608-0. [DOI] [PubMed] [Google Scholar]
  2. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  3. Bylsma N., Drakenberg T., Andersson I., Leadlay P. F., Forsén S. Prokaryotic calcium-binding protein of the calmodulin superfamily. Calcium binding to a Saccharopolyspora erythraea 20 kDa protein. FEBS Lett. 1992 Mar 24;299(1):44–47. doi: 10.1016/0014-5793(92)80096-y. [DOI] [PubMed] [Google Scholar]
  4. Clore G. M., Gronenborn A. M., Bax A. A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information. J Magn Reson. 1998 Jul;133(1):216–221. doi: 10.1006/jmre.1998.1419. [DOI] [PubMed] [Google Scholar]
  5. Clore G. M., Gronenborn A. M., Tjandra N. Direct structure refinement against residual dipolar couplings in the presence of rhombicity of unknown magnitude. J Magn Reson. 1998 Mar;131(1):159–162. doi: 10.1006/jmre.1997.1345. [DOI] [PubMed] [Google Scholar]
  6. 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 A resolution. J Mol Biol. 1993 Jan 20;229(2):461–471. doi: 10.1006/jmbi.1993.1046. [DOI] [PubMed] [Google Scholar]
  7. Cox J. A., Bairoch A. Sequence similarities in calcium-binding proteins. Nature. 1988 Feb 11;331(6156):491–491. doi: 10.1038/331491a0. [DOI] [PubMed] [Google Scholar]
  8. Craescu C. T., Prêcheur B., van Riel A., Sakamoto H., Cox J. A., Engelborghs Y. 1H and 15N resonance assignment of the calcium-bound form of the Nereis diversicolor sarcoplasmic Ca(2+)-binding protein. J Biomol NMR. 1998 Nov;12(4):565–566. doi: 10.1023/a:1008361202496. [DOI] [PubMed] [Google Scholar]
  9. Flaherty K. M., Zozulya S., Stryer L., McKay D. B. Three-dimensional structure of recoverin, a calcium sensor in vision. Cell. 1993 Nov 19;75(4):709–716. doi: 10.1016/0092-8674(93)90491-8. [DOI] [PubMed] [Google Scholar]
  10. Hermann A., Cox J. A. Sarcoplasmic calcium-binding protein. Comp Biochem Physiol B Biochem Mol Biol. 1995 Jul;111(3):337–345. doi: 10.1016/0305-0491(94)00218-j. [DOI] [PubMed] [Google Scholar]
  11. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  12. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  13. Ikura M., Kay L. E., Bax A. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry. 1990 May 15;29(19):4659–4667. doi: 10.1021/bi00471a022. [DOI] [PubMed] [Google Scholar]
  14. Ikura M., Spera S., Barbato G., Kay L. E., Krinks M., Bax A. Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy. Biochemistry. 1991 Sep 24;30(38):9216–9228. doi: 10.1021/bi00102a013. [DOI] [PubMed] [Google Scholar]
  15. Kawamura S., Hisatomi O., Kayada S., Tokunaga F., Kuo C. H. Recoverin has S-modulin activity in frog rods. J Biol Chem. 1993 Jul 15;268(20):14579–14582. [PubMed] [Google Scholar]
  16. Kay L. E., Clore G. M., Bax A., Gronenborn A. M. Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution. Science. 1990 Jul 27;249(4967):411–414. doi: 10.1126/science.2377896. [DOI] [PubMed] [Google Scholar]
  17. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  18. Kördel J., Forsén S., Chazin W. J. 1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k. Biochemistry. 1989 Aug 22;28(17):7065–7074. doi: 10.1021/bi00443a043. [DOI] [PubMed] [Google Scholar]
  19. Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [DOI] [PubMed] [Google Scholar]
  20. Moss S. E., Crumpton M. J. The lipocortins and the EF hand proteins: Ca2(+)-binding sites and evolution. Trends Biochem Sci. 1990 Jan;15(1):11–12. doi: 10.1016/0968-0004(90)90118-u. [DOI] [PubMed] [Google Scholar]
  21. Prêcheur B., Cox J. A., Petrova T., Mispelter J., Craescu C. T. Nereis sarcoplasmic Ca2+-binding protein has a highly unstructured apo state which is switched to the native state upon binding of the first Ca2+ ion. FEBS Lett. 1996 Oct 14;395(1):89–94. doi: 10.1016/0014-5793(96)01007-1. [DOI] [PubMed] [Google Scholar]
  22. Slupsky C. M., Reinach F. C., Smillie L. B., Sykes B. D. Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy. Protein Sci. 1995 Jul;4(7):1279–1290. doi: 10.1002/pro.5560040704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Swan D. G., Cortes J., Hale R. S., Leadlay P. F. Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein. J Bacteriol. 1989 Oct;171(10):5614–5619. doi: 10.1128/jb.171.10.5614-5619.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Swan D. G., Hale R. S., Dhillon N., Leadlay P. F. A bacterial calcium-binding protein homologous to calmodulin. Nature. 1987 Sep 3;329(6134):84–85. doi: 10.1038/329084a0. [DOI] [PubMed] [Google Scholar]
  25. Tjandra N., Bax A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science. 1997 Nov 7;278(5340):1111–1114. doi: 10.1126/science.278.5340.1111. [DOI] [PubMed] [Google Scholar]
  26. Tjandra N., Omichinski J. G., Gronenborn A. M., Clore G. M., Bax A. Use of dipolar 1H-15N and 1H-13C couplings in the structure determination of magnetically oriented macromolecules in solution. Nat Struct Biol. 1997 Sep;4(9):732–738. doi: 10.1038/nsb0997-732. [DOI] [PubMed] [Google Scholar]
  27. Tolman J. R., Flanagan J. M., Kennedy M. A., Prestegard J. H. Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9279–9283. doi: 10.1073/pnas.92.20.9279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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. 1992 Mar 20;224(2):413–426. doi: 10.1016/0022-2836(92)91004-9. [DOI] [PubMed] [Google Scholar]
  29. Wishart D. S., Sykes B. D., Richards F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992 Feb 18;31(6):1647–1651. doi: 10.1021/bi00121a010. [DOI] [PubMed] [Google Scholar]
  30. Wishart D. S., Sykes B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR. 1994 Mar;4(2):171–180. doi: 10.1007/BF00175245. [DOI] [PubMed] [Google Scholar]
  31. Yamagata K., Goto K., Kuo C. H., Kondo H., Miki N. Visinin: a novel calcium binding protein expressed in retinal cone cells. Neuron. 1990 Mar;4(3):469–476. doi: 10.1016/0896-6273(90)90059-o. [DOI] [PubMed] [Google Scholar]
  32. Zhang O., Kay L. E., Olivier J. P., Forman-Kay J. D. Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR. 1994 Nov;4(6):845–858. doi: 10.1007/BF00398413. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES