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
. 2000 Jan;9(1):49–52. doi: 10.1110/ps.9.1.49

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron.

A H Yang 1, R T MacGillivray 1, J Chen 1, Y Luo 1, Y Wang 1, G D Brayer 1, A B Mason 1, R C Woodworth 1, M E Murphy 1
PMCID: PMC2144434  PMID: 10739246

Abstract

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Aisen P. Transferrin, the transferrin receptor, and the uptake of iron by cells. Met Ions Biol Syst. 1998;35:585–631. [PubMed] [Google Scholar]
  2. Bailey S., Evans R. W., Garratt R. C., Gorinsky B., Hasnain S., Horsburgh C., Jhoti H., Lindley P. F., Mydin A., Sarra R. Molecular structure of serum transferrin at 3.3-A resolution. Biochemistry. 1988 Jul 26;27(15):5804–5812. doi: 10.1021/bi00415a061. [DOI] [PubMed] [Google Scholar]
  3. Baker E. N., Anderson B. F., Baker H. M., Day C. L., Haridas M., Norris G. E., Rumball S. V., Smith C. A., Thomas D. H. Three-dimensional structure of lactoferrin in various functional states. Adv Exp Med Biol. 1994;357:1–12. doi: 10.1007/978-1-4615-2548-6_1. [DOI] [PubMed] [Google Scholar]
  4. Bewley M. C., Tam B. M., Grewal J., He S., Shewry S., Murphy M. E., Mason A. B., Woodworth R. C., Baker E. N., MacGillivray R. T. X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32. Biochemistry. 1999 Feb 23;38(8):2535–2541. doi: 10.1021/bi9824543. [DOI] [PubMed] [Google Scholar]
  5. Dewan J. C., Mikami B., Hirose M., Sacchettini J. C. Structural evidence for a pH-sensitive dilysine trigger in the hen ovotransferrin N-lobe: implications for transferrin iron release. Biochemistry. 1993 Nov 16;32(45):11963–11968. doi: 10.1021/bi00096a004. [DOI] [PubMed] [Google Scholar]
  6. Funk W. D., MacGillivray R. T., Mason A. B., Brown S. A., Woodworth R. C. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein. Biochemistry. 1990 Feb 13;29(6):1654–1660. doi: 10.1021/bi00458a043. [DOI] [PubMed] [Google Scholar]
  7. He Q. Y., Mason A. B., Tam B. M., MacGillivray R. T., Woodworth R. C. Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site. Biochemistry. 1999 Jul 27;38(30):9704–9711. doi: 10.1021/bi990134t. [DOI] [PubMed] [Google Scholar]
  8. He Q. Y., Mason A. B., Woodworth R. C., Tam B. M., MacGillivray R. T., Grady J. K., Chasteen N. D. Inequivalence of the two tyrosine ligands in the N-lobe of human serum transferrin. Biochemistry. 1997 Dec 2;36(48):14853–14860. doi: 10.1021/bi9719556. [DOI] [PubMed] [Google Scholar]
  9. He Q. Y., Mason A. B., Woodworth R. C., Tam B. M., MacGillivray R. T., Grady J. K., Chasteen N. D. Mutations at nonliganding residues Tyr-85 and Glu-83 in the N-lobe of human serum transferrin. Functional second shell effects. J Biol Chem. 1998 Jul 3;273(27):17018–17024. doi: 10.1074/jbc.273.27.17018. [DOI] [PubMed] [Google Scholar]
  10. He Q. Y., Mason A. B., Woodworth R. C., Tam B. M., Wadsworth T., MacGillivray R. T. Effects of mutations of aspartic acid 63 on the metal-binding properties of the recombinant N-lobe of human serum transferrin. Biochemistry. 1997 May 6;36(18):5522–5528. doi: 10.1021/bi963028p. [DOI] [PubMed] [Google Scholar]
  11. Jeffrey P. D., Bewley M. C., MacGillivray R. T., Mason A. B., Woodworth R. C., Baker E. N. Ligand-induced conformational change in transferrins: crystal structure of the open form of the N-terminal half-molecule of human transferrin. Biochemistry. 1998 Oct 6;37(40):13978–13986. doi: 10.1021/bi9812064. [DOI] [PubMed] [Google Scholar]
  12. Li Y., Harris W. R., Maxwell A., MacGillivray R. T., Brown T. Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins. Biochemistry. 1998 Oct 6;37(40):14157–14166. doi: 10.1021/bi9810454. [DOI] [PubMed] [Google Scholar]
  13. Lin L. N., Mason A. B., Woodworth R. C., Brandts J. F. Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site. Biochem J. 1993 Jul 15;293(Pt 2):517–522. doi: 10.1042/bj2930517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. MacGillivray R. T., Moore S. A., Chen J., Anderson B. F., Baker H., Luo Y., Bewley M., Smith C. A., Murphy M. E., Wang Y. Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release. Biochemistry. 1998 Jun 2;37(22):7919–7928. doi: 10.1021/bi980355j. [DOI] [PubMed] [Google Scholar]
  15. Mason A. B., Woodworth R. C., Oliver R. W., Green B. N., Lin L. N., Brandts J. F., Tam B. M., Maxwell A., MacGillivray R. T. Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 1996 Aug;8(1):119–125. doi: 10.1006/prep.1996.0081. [DOI] [PubMed] [Google Scholar]
  16. Mason A., He Q. Y., Tam B., MacGillivray R. A., Woodworth R. Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe-lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies. Biochem J. 1998 Feb 15;330(Pt 1):35–40. doi: 10.1042/bj3300035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  18. Steinlein L. M., Graf T. N., Ikeda R. A. Production and purification of N-terminal half-transferrin in Pichia pastoris. Protein Expr Purif. 1995 Oct;6(5):619–624. doi: 10.1006/prep.1995.1081. [DOI] [PubMed] [Google Scholar]
  19. Steinlein L. M., Ligman C. M., Kessler S., Ikeda R. A. Iron release is reduced by mutations of lysines 206 and 296 in recombinant N-terminal half-transferrin. Biochemistry. 1998 Sep 29;37(39):13696–13703. doi: 10.1021/bi980318s. [DOI] [PubMed] [Google Scholar]
  20. Wang Y., Chen J., Luo Y., Funk W. D., Mason A. B., Woodworth R. C., MacGillivray R. T., Brayer G. D. Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin. J Mol Biol. 1992 Sep 20;227(2):575–576. doi: 10.1016/0022-2836(92)90910-c. [DOI] [PubMed] [Google Scholar]
  21. Woodworth R. C., Mason A. B., Funk W. D., MacGillivray R. T. Expression and initial characterization of five site-directed mutants of the N-terminal half-molecule of human transferrin. Biochemistry. 1991 Nov 12;30(45):10824–10829. doi: 10.1021/bi00109a002. [DOI] [PubMed] [Google Scholar]
  22. Zak O., Aisen P., Crawley J. B., Joannou C. L., Patel K. J., Rafiq M., Evans R. W. Iron release from recombinant N-lobe and mutants of human transferrin. Biochemistry. 1995 Nov 7;34(44):14428–14434. doi: 10.1021/bi00044a020. [DOI] [PubMed] [Google Scholar]
  23. Zak O., Tam B., MacGillivray R. T., Aisen P. A kinetically active site in the C-lobe of human transferrin. Biochemistry. 1997 Sep 9;36(36):11036–11043. doi: 10.1021/bi970628v. [DOI] [PubMed] [Google Scholar]

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

RESOURCES