Lactoferrin anion sites

Lactobacillus plantarum, 33 214 Lactoferrin, 41 390 anion sites, 41 418 biological role, 41 392-393... 

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). 

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. 

M. Hartdas, G. E. Norris. S. V. Rumball, and C. A. Smith, "Metal and Anion Binding Sites in Lactoferrin and Related Proteins, " Pure Appl Chem. 1990,62, 1067.]... 

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.

Fig. 16. Comparison of the anion binding sites in lactoferrin (left) and the bacterial sulfate-binding protein (right). From Baker et al. (82), with permission.

Fig. 21. Metal and anion binding in the crystal structure of Cu2+-substituted human lactoferrin. (a) The five-coordinate N-lobe copper site, in which either the anion or Tyr 92 may be protonated, and (b) the six-coordinate C-lobe copper site. In each case the axial ligands, Asp 60 and Tyr 192 in the N-lobe and Asp 395 and Tyr 528 in the C-lobe, are present but not shown. Adapted from Shongwe et al. (192), with permission.

Pig. 27. Possible sites for the binding of secondary, nonsynergistic anions, which may perturb the iron site and modulate release. Residue 121 is the essential arginine. Residues 210 and 301 are located behind the iron site, near the Tyr ligands and the hinge region. Numbering is as for the N-lobe of lactoferrin. The identities of these residues, which vary in the two lobes and between different transferrins, can be seen in Table III. 

The four metal-binding amino acid residues (2 Tyr, 1 Asp, 1 His) are present in both N- and C-sites of all transferrins so far sequenced, apart from melanotransferrin and the insect proteins (Table III). The same is true of the anion-binding Arg and Thr residues, and the residues at the N-terminus of the anion-binding helix are also strongly conserved. Superposition of the 81 common atoms of these residues, plus metal and anion, shows that their rms deviation in the N- and C-sites of diferric human lactoferrin is only 0.3 A. This close structural similarity is reflected in their spectroscopic properties. Where these have been compared, with the physiological Fe3+ and C032- ions bound, they are so similar as to be virtually identical (107, 56, 199). Nevertheless, there are a number of factors that can potentially lead to inequivalence in properties ... 

Distinct differences are also seen when anions other than C032 are used. The crystal structure of oxalate-substituted diferric lactoferrin shows differences in the anion binding in the two sites in the C-site the oxalate is symmetric bidentate, whereas in the N-site it is asymmetric (193). When Cu2+ is the metal ion the oxalate binding differences become even more pronounced. Copper-transferrin binds oxalate only in its N-terminal site (91). Copper-lactoferrin and copper-ovotransfer-rin each bind two oxalate ions but binding occurs preferentially in the C-lobe (157,192). These different affinities mean that hybrid complexes can be prepared with oxalate in one site and carbonate in the other (92, 157, 192). The use of oxalate as synergistic anion gives rise to spectroscopically distinct sites for other metal ions also (171). 

The above mechanism is totally consistent with the crystallographic results from the various forms of lactoferrin and transferrin (Section III.B). These lead to a structural model of binding shown pictorially in Fig. 29. In the first step the synergistic anion (usually carbonate) is bound in the specific site on domain 2 of each lobe. Binding may be preceded by electrostatic attraction from the exposed helix N-termini and several basic sidechains in the open interdomain cleft. 

Several proteins that exist in food (e.g., lactoferrin, ferritin, transferritin, heme protein) possess strong binding sites for iron. Reducing agents (ascorbate, cysteine, superoxide anion) to low pH causes release of iron from proteins and accelerates lipid oxidation (34). Some amino acids and peptides found in muscle foods (e.g., carnosine) are capable of chelating metal ions and inhibit their prooxidant activity (35, 36). 

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