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:

Key residues involved in calcium-binding motifs in EGF-like domains

Abstract

MANY extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a β/-hydroxylated residue)1. These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues2. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein3. Similar results have been obtained for bovine factor X4. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift3 in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gin 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations5 in this domain, Asp 47→ Glu, and Asp 64 → Asn, present in patients with haemophilia B, reduce calcium binding to the domain >4-fold and >1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gin 50 → Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs1,6–8.

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

Similar content being viewed by others

References

  1. Rees, D. J. G. et al. EMBO J. 7, 2053–2061 (1988).

    Article  CAS  Google Scholar 

  2. Furie, B. & Furie, B. C. Cell 53, 505–518 (1988).

    Article  CAS  Google Scholar 

  3. Handford, P. A. et el. EMBO J. 9, 475–480 (1990).

    Article  CAS  Google Scholar 

  4. Persson, E. et al. J. biol. Chem. 264, 16897–16904 (1989).

    CAS  PubMed  Google Scholar 

  5. Winship, P. & Dragon, A. C. Br. J. Haematol 77, 102–109 (1991).

    Article  CAS  Google Scholar 

  6. Wharton, K. A., Johansen, K. M., Xu, T. & Artavanis-Tsakonas, S. Cell 43, 567–581 (1985).

    Article  CAS  Google Scholar 

  7. Vässin, H., Bremer, K. A., Knust, E. & Campos-Ortega, J. EMBO J. 6, 3431–3440 (1987).

    Article  Google Scholar 

  8. Tepass, U., Theres, C. & Knust, E. Cell 61, 787–799 (1990).

    Article  CAS  Google Scholar 

  9. Cooke, R. M. et al. Nature 327, 339–341 (1987).

    Article  ADS  CAS  Google Scholar 

  10. Baron, M. thesis, Univ. Oxford (1990).

  11. Morita, T. & Kisiel, W. Biochem. biophys. Res. Commun. 130, 841–847 (1985).

    Article  CAS  Google Scholar 

  12. Fehon, R. G. et al. Cell 61, 523–534 (1990).

    Article  CAS  Google Scholar 

  13. Öhlin, A. et al. J. biol. Chem. 263, 19240–19248 (1988).

    PubMed  Google Scholar 

  14. Kurosawa, S., Stearns, D. J., Jackson, K. W. & Esmon, C. T. J. biol. Chem. 263, 5993–5996 (1988).

    CAS  PubMed  Google Scholar 

  15. Villiers, C. L., Arlaud, G. J. & Colomb, M. G. Proc. natn. Acad. Sci. U.S.A. 82, 4477–4481 (1985).

    Article  ADS  CAS  Google Scholar 

  16. Kunkel, T. Proc. natn. Acad. Sci. U.S.A. 82, 488–492 (1985).

    Article  ADS  CAS  Google Scholar 

  17. Saiki, R. K. et al. Science 239, 487–491 (1988).

    Article  ADS  CAS  Google Scholar 

  18. Yoshitake, S., Schach, B. G., Foster, D. C., Davie, E. W. & Kurachi, K. Biochemistry 24, 3736–3750 (1985).

    Article  CAS  Google Scholar 

  19. Winship, P. R. Nucleic Acids Res. 17, 1266 (1989).

    Article  CAS  Google Scholar 

  20. Jaenicke, R. & Rudolph, R. in Protein Structure: A Practical Approach (ed. Creighton, T. E.) 208–209 (IRL, Oxford, 1989).

    Google Scholar 

  21. Fernlund, P. & Stenflo, J. J. biol. Chem. 258, 12509–12512 (1983).

    CAS  Google Scholar 

  22. Nishimura, H. et al. Thromb. Haem. 62, 17 (1989).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Handford, P., Mayhew, M., Baron, M. et al. Key residues involved in calcium-binding motifs in EGF-like domains. Nature 351, 164–167 (1991). https://doi.org/10.1038/351164a0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/351164a0

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

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