“Dilysine Trigger” in Transferrins Probed by Mutagenesis of Lactoferrin:  Crystal Structures of the R210G, R210E, and R210L Mutants of Human Lactoferrin

The mammalian iron-binding proteins lactoferrin (Lf) and transferrin (Tf) bind iron very tightly, but reversibly. Despite homologous structures and essentially identical iron binding sites, Tf begins to release iron at pH 6.0, whereas Lf retains iron to pH ∼3.5. This difference in iron retention giv...

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Published in:Biochemistry (Easton) 2002-12, Vol.41 (48), p.14167-14175
Main Authors: Peterson, Neil A, Arcus, Vickery L, Anderson, Bryan F, Tweedie, John W, Jameson, Geoffrey B, Baker, Edward N
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Animals
Arginine - genetics
Binding Sites - genetics
Cell Line
Cricetinae
Crystallization
Crystallography, X-Ray
Dipeptides - chemistry
Dipeptides - metabolism
Glutamic Acid - genetics
Glycine - genetics
Humans
Iron - metabolism
Lactoferrin - chemistry
Lactoferrin - genetics
Lactoferrin - metabolism
Leucine - genetics
Mutagenesis, Site-Directed
Protein Structure, Tertiary - genetics
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Transferrin - chemistry
Transferrin - metabolism
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Summary:The mammalian iron-binding proteins lactoferrin (Lf) and transferrin (Tf) bind iron very tightly, but reversibly. Despite homologous structures and essentially identical iron binding sites, Tf begins to release iron at pH 6.0, whereas Lf retains iron to pH ∼3.5. This difference in iron retention gives the two proteins different biological roles. Two lysine residues, Lys 206 and Lys 296, which form a hydrogen-bonded dilysine pair in human Tf, have been shown to strongly influence iron release from the N-lobe. The equivalent residues in human Lf are Arg 210 and Lys 301, and we have here mutated Arg 210 in the N-lobe half-molecule of human lactoferrin, LfN, to probe its role in iron release. The LfN mutants R210G, R210E, and R210L were expressed, purified, and crystallized, and their crystal structures were determined and refined at resolutions of 1.95 Å (R210G), 2.2 Å (R210E), and 2.0 Å (R210L). The overall structures are very similar to that of wild-type LfN, but with small differences in domain orientations. In each of the mutants, however, Lys 301 (equivalent to Lys 296 in Tf) changes its conformation to fill the space occupied by Arg 210 Nη2 in wild-type LfN, interacting with the two tyrosine ligands Tyr 92 and Tyr 192. By comparison with other Lf and Tf structures, we conclude that Lys 301 (or Lys 296 in Tf) only occupies this site when residue 210 (206 in Tf) is nonpositive (neutral as in R210G and R210L or negative as in R210E). Thus, Lys 206 in the Tf dilysine pair is identified as having a depressed pK a. Three specific sites are variably occupied by polar groups in the Lf mutants and other Lf and Tf proteins, and when coupled with iron-release data, these give new insights into the factors that most influence iron retention at low pH.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi020443a