Structure of copper- and oxalate-substituted human lactoferrin at 2.0 Å resolution

The three-dimensional structure of human dicupric monooxalate lactoferrin, Cu₂oxLf, has been determined to 2.0 Å resolution, using X-ray diffraction data collected by diffractometry to 2.5 Å resolution, and oscillation photography on a synchrotron source to 2.0 Å resolution. Difference electron-density maps calculated between Cu₂oxLf and both dicupric lactoferrin, Cu₂Lf, and diferric lactoferrin, Fe₂Lf, showed that the oxalate had replaced a carbonate in the C-terminal binding site, and that, relative to Cu₂Lf, there were no significant differences in the N-terminal site. The structure was then refined crystallographically by restrained least-squares methods. The final model, in which the r.m.s. deviation in bond distances is 0.017 Å, contains 5314 protein atoms (691 residues), two Cu²⁺ ions, one bicarbonate ion, one oxalate ion, 325 solvent molecules and one sugar residue. The crystallographic R factor of 0.193 is for 46 134 reflections in the range 8.0 to 2.0 Å resolution. The oxalate ion is coordinated to copper in a 1,2-bidentate fashion, and the added bulk of the anion results in the rearrangement of the side chains of nearby arginine and tyrosine residues. No other major alterations in the molecule can be observed, the overall protein structure being the same as that for Cu₂Lf and Fe₂Lf.