Proteins of the transferrin family bind, with high affinity, two Fe3+ ions and two CO32- ions but can also bind other metal ions and other anions. In order to find out how the protein structure and its two binding sites adapt to the binding of larger anions, we have determined the crystal structure of oxalate-substituted diferric lactoferrin at 2.4 Å resolution. The final model has a crystallographic R-factor of 0.196 for all data in the range 8.0-2.4 Å. Substitution of oxalate for carbonate does not produce any significant change in the polypeptide folding or domain closure. Both binding sites are perturbed, however, and the effects are different in each. In the C-lobe site the oxalate ion is bound to iron in symmetric 1,2-bidentate fashion whereas in the N-lobe the anion coordination is markedly asymmetric. The difference arises because in each site substitution of the larger anion causes displacement of the arginine that forms one wall of the anion binding site: the movement is different in each case, however, because of different interactions with 'second shell' amine acid residues in the binding cleft. These observations provide an explanation for the site inequivalences that accompany the substitution of non-native anions and cations.
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