ANION-BINDING BY TRANSFERRINS - IMPORTANCE OF 2ND-SHELL EFFECTS REVEALED BY THE CRYSTAL-STRUCTURE OF OXALATE-SUBSTITUTED DIFERRIC LACTOFERRIN

Proteins of the transferrin family bind, with high affinity, two Fe3ions and two CO32- ions can also bind other metal ions and other anion s. 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 cr ystal structure of oxalate-substituted diferric lactoferrin at 2.4 Ang strom resolution. The final model has a crystallographic R-factor of 0 .196 for all data in the range 8.0-2.4 Angstrom. Substitution of oxala te for carbonate does not produce any significant change in the polype ptide folding or domain closure. Both binding sites are perturbed, how ever, and the effects are different in each. In the C-lobe site the ox alate ion is bound to iron in symmetric 1,2-bidentate fashion whereas in the N-lobe the anion coordination is markedly asymmetric. The diffe rence arises because in each site substitution of the larger anion cau ses displacement of the arginine that forms one wall of the anion bind ing site; the movement is different in each case, however, because of different interactions with ''second shell'' amino acid residues in th e binding cleft. These observations provide an explanation for the sit e inequivalences that accompany the substitution of non-native anions and cations.