Abstract
This is the first protein intermediate obtained in the crystalline state by the simultaneous process of Fe(3+) binding and crystal nucleation and is also the first structure of an intermediate of lactoferrin in the Fe(3+) binding pathway. Lactoferrin is an iron-binding 80-kDa glycoprotein. It binds Fe(3+) very tightly in a closed interdomain cleft in both lobes. The iron-free structure of lactoferrin, on the other hand, adopts an open conformation with domains moving widely apart. These studies imply that initial Fe(3+) binding must be in the open form. The protein intermediate was crystallized by the microdialysis method. The protein solution, with a concentration of 100 mg/ml in 10 mm Tris-HCl, pH 8.0, was loaded in a capillary and dialyzed against the same buffer containing 26% (v/v) ethanol placed in a reservoir. FeCl(3) and CO(3)(2-) in excess molar ratios to that of protein in its solution were added to the reservoir buffer. The crystals appeared after some hours and grew to the optimum size within 36 h. The structure was determined by molecular replacement method and refined to final R- and R-free factors of 0.187 and 0.255, respectively. The present structure showed that the protein molecule adopts an open conformation similar to that of camel apolactoferrin. The electron density map clearly indicated the presence of two iron atoms, one in each lobe with 4-fold coordinations: two by the protein ligands of Tyr-92(433) OH and Tyr-192(526) OH and two other coordination sites occupied by oxygen atoms of bidentate CO(3)(2-) ions leading to a tetrahedral intermediate. The CO(3)(2-) anion is stabilized through hydrogen bonds with the synergistic anion-binding site Arg-121(463) and with Ser-122 Ogamma in the N-lobe and Thr-464 Ogamma in C-lobe. The third oxygen atom of CO(3)(2-) interacts with a water molecule in both lobes.
Highlights
This is the first protein intermediate obtained in the crystalline state by the simultaneous process of Fe3؉ binding and crystal nucleation and is the first structure of an intermediate of lactoferrin in the Fe3؉ binding pathway
The electron density map clearly indicated the presence of two iron atoms, one in each lobe with 4-fold coordinations: two by the protein ligands of Tyr-92(433) OH and Tyr-192(526) OH and two other coordination sites occupied by oxygen atoms of bidentate CO32؊ ions leading to a tetrahedral intermediate
The current crystal structure corresponds to intermediate III, LF1⁄72 Fe3ϩ1⁄72 CO32Ϫ complex (Fig. 1), with an open conformation in which only four of the six Fe3ϩ coordination sites are occupied by the side chains of protein ligands Tyr-92(433) and Tyr-192(526) and bidentate CO32Ϫ
Summary
CRYSTAL STRUCTURE OF AN IRON-SATURATED INTERMEDIATE IN THE Fe3ϩ BINDING PATHWAY OF CAMEL LACTOFERRIN AT 2.7 Å RESOLUTION*. The iron-free structure of lactoferrin, on the other hand, adopts an open conformation with domains moving widely apart. These studies imply that initial Fe3؉ binding must be in the open form. The crystal structures of fully ironsaturated differic forms of lactoferrins [4, 5, 11,12,13] have revealed that the two domains of each lobe are closed over an Fe3ϩ ion. The four protein ligands have different locations, and the iron-loaded open form is a possible transient intermediate toward the fully saturated holoform with closed domains. The structure of an iron-saturated open form of the N-lobe of hen ovotransferrin with an unnatural anion nitrilotriacetate has been reported recently [15]
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