Crystal structure and iron-binding properties of the R210K mutant of the N-lobe of human lactoferrin: implications for iron release from transferrins.
Lactoferrin (Lf) and serum transferrin (Tf) combine high-affinity iron binding with an ability to release this iron at reduced pH. Lf, however, retains iron to significantly lower pH than Tf, giving the two proteins distinct functional roles. In this paper, we compared the iron-release profiles for human Lf, Tf, and their N-lobe half-molecules Lf(N) and Tf(N) and showed that half of the difference in iron retention at low pH ( approximately 1.3 pH units) results from interlobe interactions in Lf. To probe factors intrinsic to the N-lobes, we further examined the specific role of two basic residues that are proposed to form a pH-sensitive dilysine trigger for iron release in the N-lobe of Tf [Dewan, J. C., Mikami, B., Hirose, M., and Sacchettini, J. C. (1993) Biochemistry 32, 11963-11968] by mutating Arg 210 to Lys in the N-lobe half-molecule Lf(N). The R210K mutant was expressed, purified, and crystallized, and its crystal structure was determined and refined at 2.0-A resolution to a final R factor (R(free)) of 19.8% (25.0%). The structure showed that Lys 210 and Lys 301 in R210K do not form a dilysine interaction like that between Lys 206 and Lys 296 in human Tf. The R210K mutant retained iron to lower pH than Tf(N), consistent with the absence of the dilysine interaction but released iron at approximately 0.7 pH units higher than Lf(N). We conclude that (i) the ability of Lf to retain iron to significantly lower pH than Tf is due equally to interlobe interactions and to the absence in Lfs of an interaction analogous to the dilysine pair in Tfs, even when two lysines are present at the corresponding sequence positions, and (ii) an appropriately positioned basic residue (Arg 210 in human Lf) modulates iron release by inhibiting protonation of the N-lobe iron ligands, specifically His 253.
Peterson
N A
NA
Institute of Molecular Biosciences and Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Anderson
B F
BF
Jameson
G B
GB
Tweedie
J W
JW
Baker
E N
EN
D001665:Binding Sites; D002254:Carbonates / Q000737:chemistry; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007781:Lactoferrin / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014168:Transferrin / Q000737:chemistry*
D002254:Carbonates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron; D007781:Lactoferrin; D008239:Lysine