Lactoferrin pp 25-38 | Cite as

Altered Domain Closure and Iron Binding in Lactoferrin Mutants

  • H. Rick Faber
  • Bryan F. Anderson
  • Heather M. Baker
  • Tony Bland
  • Catherine L. Day
  • Hale Nicholson
  • Steven Shewry
  • John W. Tweedie
  • Edward N. Baker
Part of the Experimental Biology and Medicine book series (EBAM, volume 28)

Summary

Two features of the functional properties of lactoferrin are its ability to bind iron exceptionally tightly and the coupling of rigid-body domain movements to iron binding and release. The latter cause transitions between open and closed forms of the protein. Using site-directed mutagenesis and X-ray crystallography we have examined the importance of selected residues, including the iron ligands Asp 60 and His 253, the anion-binding Arg 121, and Pro 251 in the hinge region. Five mutants, D60S, R121S, R121E, H253M, and P251 A, have been prepared in the context of the N-terminal half-molecule of human lactoferrin, Lfn, and three-dimensional structures have been determined in each case. In D60S the mutation leads to weakened iron binding because a water molecule binds to the iron atom in place of Asp 60. Interdomain interactions are also weakened, and the loss of the Asp side-chain causes a significant change in domain closure; the domains move closer together by 7° in the mutant. The R121S and R121E mutants show altered anion binding and very small changes in domain orientations. The H253M and P251A mutants show identical domain closure to wild-type LfN, but the iron site is altered in H253M; the Met 253 side-chain is not bound to iron, leaving a 5-coordinate site. These results are interpreted in terms of the roles of each of the residues in iron binding and release.

Keywords

Crystallization Filtration Hydroxyl Carboxylate Polypeptide 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., and Baker, E. N. (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 Å resolution. J. Mol. Biol. 209, 711–734.CrossRefGoogle Scholar
  2. Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V., and Baker, E. N. (1990) Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins. Nature (Lond.) 344, 784–787.CrossRefGoogle Scholar
  3. Baker, E. N., Baker, H. M., Smith, C. A., Stebbins, M. R., Kahn, M., Hellström, K. E., and Hellström, I. (1992) Human melanotransferrin (p97) has only one functional iron-binding site. FEBS Lett. 298, 215–219.CrossRefGoogle Scholar
  4. Baker, E. N. (1994) Structure and reactivity of transferrins. Adv. Inorg. Chem. 41, 389–463.CrossRefGoogle Scholar
  5. Baker, H. M., Day, C. L., Norris, G. E., and Baker, E. N. (1994) Enzymatic deglycosylation as a tool for crystallization of mammalian binding proteins. Acta Crystallogr. Sect. D 50, 380–384.CrossRefGoogle Scholar
  6. Day, C. L., Norris, G. E., Anderson, B. R, Tweedie, J. W., and Baker, E. N. (1992a) Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms. J. Mol. Biol. 228, 973–974.CrossRefGoogle Scholar
  7. Day, C. L., Stowell, K. M., Baker, E. N., and Tweedie, J. W. (1992b) Studies of the N-terminal half of human lactoferrin from the cloned cDNA demonstrate that interlobe interactions modulate iron release. J. Biol. Chem. 267, 13,857–13,862.Google Scholar
  8. Day, C. L., Anderson, B. R, Tweedie, J. W., and Baker, E. N. (1993) Structure of the recombinant N-terminal lobe of human lactoferrin at 2.0 Å resolution. J. Mol. Biol. 232, 1084–1100.CrossRefGoogle Scholar
  9. Faber, H. R., Anderson, B. R, Baker, H. M., and Baker, E. N. (1995a) The crystal structure of a fully-open form of human apolactoferrin. Manuscript in preparation.Google Scholar
  10. Faber, H. R., Bland, T., Day, C. L., Norris, G. E., Tweedie, J. W., and Baker, E. N. (1995b) Altered domain closure and iron binding in transferrins: crystal structure of the Asp60Ser mutant of the amino-terminal half-molecule of human lactoferrin. J. Mol. Biol, in press.Google Scholar
  11. Gerstein, M., Anderson, B. R, Norris, G. E., Baker, E. N., Lesk, A. M., and Chothia, C. (1993) Domain closure in lactoferrin. J. Mol Biol. 234, 357–372.CrossRefGoogle Scholar
  12. Grossmann, J. G., Mason, A. B., Woodworth, R. C., Neu, M., Lindley, P. R, and Hasnain, S. S. (1993) Asp ligand provides the trigger for closure of transferrin molecules. J. Mol Biol. 231, 554–558.CrossRefGoogle Scholar
  13. Haridas, M., Anderson, B. R, and Baker, E. N. (1995) The structure of human diferric lactoferrin, refined at 2.2 Å resolution. Acta Crystallogr. Sect. D 51, 629–646.CrossRefGoogle Scholar
  14. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Meth. Enzymol. 154, 367–382.CrossRefGoogle Scholar
  15. Rochard, E., Legrand, D., Mazurier, J., Montreuil, J., and Spik, G. (1989) The N-terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin-stimulated peripheral blood human lymphocyte receptors. FEBS Lett. 255, 201–204.CrossRefGoogle Scholar
  16. Tronrud, D. E., Ten Eyck, L. E, and Matthews, B. W. (1987) An efficient general-purpose least-squares refinement program for macromolecular structures. Acta Crystallogr. Sect. A 43, 489–501.CrossRefGoogle Scholar
  17. Tweedie, J. W., Bain, H. B., Day, C. L., Nicholson, H. H., Mead, P. E., Sheth, B., and Stowell, K. M. (1994) Lactoferrin cDNA: expression and in vitro mutagenesis. Adv. Exp. Med. Biol. 357, 197–208.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • H. Rick Faber
  • Bryan F. Anderson
  • Heather M. Baker
  • Tony Bland
  • Catherine L. Day
  • Hale Nicholson
  • Steven Shewry
  • John W. Tweedie
  • Edward N. Baker

There are no affiliations available

Personalised recommendations