Mutagenesis of the histidine ligand in human lactoferrin: iron binding properties and crystal structure of the histidine-253-->methionine mutant

Biochemistry
H NicholsonE N Baker

Abstract

The contribution of the conserved His ligand to iron binding in transferrins has been addressed by site-directed mutagenesis and X-ray crystallographic analysis. His 253 in the N-terminal half-molecule of human lactoferrin, LfN (residues 1-333), has been changed to Gly, Ala, Pro, Thr, Leu, Phe, Met, Tyr, Glu, Gln, and Cys by oligonucleotide-directed mutagenesis. The proteins have been expressed in baby hamster kidney cells, at high levels, and purified. The results show that the His ligand is essential for the stability of the iron binding site. All of the substitutions destabilized iron binding irrespective of whether the replacements were potential iron ligands or not. Iron was lost below pH approximately 6 for the Cys, Glu, and Tyr mutants and below pH 7 or higher for the others, compared with pH 5.0 for LfN. The destabilization is attributed to both steric and electronic effects. The importance of electronic effects has been shown by the crystal structure of the H253M mutant, which has been determined at an effective resolution of 2.5 A and refined to a final R factor of 0.173. The iron atom is changed from six-coordinate to five-coordinate; the Met 253 side chain is not bound to iron even though there appears to be no ster...Continue Reading

References

Jan 1, 1984·Progress in Biophysics and Molecular Biology·E N Baker, R E Hubbard
Nov 24, 1995·Journal of Molecular Biology·H KurokawaM Hirose
Jul 1, 1994·Acta Crystallographica. Section D, Biological Crystallography·H M BakerE N Baker

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Citations

Oct 3, 2002·Protein Expression and Purification·M ParamasivamA Srinivasan
Jun 3, 2009·The Pharmacogenomics Journal·L LitB Wong
Jan 28, 2003·Journal of Pediatric Gastroenterology and Nutrition·Yasushi A SuzukiBo Lönnerdal
Aug 17, 2000·Bioscience, Biotechnology, and Biochemistry·M Hirose
Nov 13, 2007·Journal of Food Science·Gen-Hung ChenShann-Tzong Jiang
Jan 22, 2013·Journal of Food Science·Suzanne M Jervis, MaryAnne Drake
Aug 23, 2005·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Lisa A LambertAnne B Mason
Aug 31, 2010·The Biochemical Journal·Husain K KhambatiAnthony B Schryvers
Nov 28, 2002·Molecular Cell·Jun YangJonathan Barasch
Feb 12, 1998·Journal of Molecular Biology·S A MooreE N Baker
Feb 27, 2015·Journal of Agricultural and Food Chemistry·Ian B O'LoughlinAndre Brodkorb

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