Transferrin anion-binding site

Early experimental evidence in support of the hypothesis that an attack on the anion is at the heart of the iron-exchange mechanism (53) was soon corroborated by work from several laboratories (54, 55, 88). Replacing carbonate with oxalate at the specific anion-binding site of transferrin results in a relatively stable ternary Fe(III)-transferrin-oxalate complex. Over the time course of many hours or even days the oxalate complex slowly reverts to the physiologic Fe(III)-transferrin-carbonate form, but since in vitro studies seldom require more than an hour or two, the biologic properties of the oxalate complex can be tested. 

Figure 19. A model for the anion- and iron-binding sites of transferrin depicted assuming an interlocking-site hypothesis. The protein furnishes five ligands to the metal in the iron binding site three tyrosines and two histidines. The carbonate ion binds to an arginine in the anion-binding site and functions as a sixth ligand to the metal center. The carbonate forms a bridge between the metal- and the anion-binding sites in the active center (36).

Fig. 7. Schematic diagram of the characteristic transferrin metal and anion binding site. Numbering is as for the N-lobe of human lactoferrin, but the same arrangement of ligands is found in the C-lobe and in the N- and C-lobes of almost all transferrins (Table III). For reference, the residue numbers for human lactoferrin and human transferrin are shown in the inset.

The bilobal structure of transferrins means that half-molecules, representing either the N-terminal or C-terminal lobe, can be relatively easily prepared, either by limited proteolysis or by recombinant DNA methods (Section III.A). Relatively high-resolution crystal structures have been determined for three such half-molecules, the proteolytic N-lobes of rabbit transferrin (74) and chicken ovotransferrin (77) at 2.3 A and the recombinant N-lobe of human lactoferrin at 2.0-A resolution (75). These show that both the protein structure and the metal and anion binding sites are the same as in the intact parent structures. In fact comparison of the metal and anion sites of the lactoferrin and transferrin half-molecules with each other and with the N-lobe of lactoferrin shows very close correspondence 92 atoms from the nine residues, plus metal and anion, making up the immediate binding site can be superimposed with an rms deviation of only 0.4 A (75). 

Harris, D. C., Gray, G. A., and Aisen, P. (1974)./. Biol. Chem. 249, 5261. 13C Nuclear Magnetic Resonance Study of the Spatial Relation of the Metal- and Anion-binding Sites of Human Transferrin. 

The iron-binding sites have been characterized by crystallographic studies on several transferrins, and in Figure 5.7 (Plate 7) that of the N-lobe of human lactoferrin is presented. The 3+ charge on the ferric ion is matched by the three anionic ligands Asp-63, Tyr-95 and Tyr-188 (the fourth, His-249, is neutral), while the charge on the carbonate anion is almost matched by the positive charge on Arg-124 and the... 

Detailed pictures of the iron-binding sites in transferrins have been provided by the crystal structures of lactoferrin (Anderson et ai, 1987, 1989 Baker etai, 1987) and serum transferrin (Bailey etal., 1988). Each structure is organized into two lobes of similar structure (the amino- and carboxy-terminal lobes) that exhibit internal sequence homology. Each lobe, in turn, is organized into two domains separated by a cleft (Fig. 3 and 10). The domains have similar folding patterns of the a//3 type. One iron site is present in each lobe, which occupies equivalent positions in the interdomain cleft. The same sets of residues serve as iron ligands to the two sites two tyrosines, one histidine, and one aspartate. Additional extra density completes the octahedral coordination of the iron and presumably corresponds to an anion and/or bound water. The iron sites are buried about 10 A below the protein surface and are inaccessible to solvent. 

The coupling between iron and anion binding is a critical component of transferrin function. In the absence of anion, transferrins bind Fe " weakly and nonspecifically. The putative anion site identified crystal-lographically is in an electrostatically positive region, adjacent to the side chain of an arginine and the amino terminus of an a helix. The anion site does not bridge the two domains, and so does not function directly as a latch that closes the cleft around the iron. It is possible that the anion may partially compensate for the presence of several basic residues in the cleft that enhance iron binding. 

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