| Abstract
| - The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferric iron. Eachof the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. Humanserum transferrin (hTF) serves as the iron transport protein in the blood; circulating transferrin binds toreceptors on the cell surface, and the complex is internalized by endocytosis. Within the cell, a reductionin pH leads to iron release from hTF in a receptor-dependent process resulting in a large conformationalchange in each lobe. In the hTF N-lobe, two critical lysines facilitate this pH-dependent conformationalchange allowing entry of a chelator to capture the iron. In the C-lobe, the lysine pair is replaced by a triadof residues: Lys534, Arg632, and Asp634. Previous studies show that mutation of any of these triadresidues to alanine results in significant retardation of iron release at both pH 7.4 and pH 5.6. In thepresent work, the role of the three residues is probed further by conversion to the residues observed at theequivalent positions in ovotransferrin (Q-K-L) and human lactoferrin (K-N-N) as well as a triad with aninterchanged lysine and arginine (K534R/R632K). As expected, all of the constructs bind iron and associatewith the receptor with nearly the same KD as the wild-type monoferric hTF control. However, interestingdifferences in the effect of the substitutions on the iron release rate in the presence and absence of thereceptor at pH 5.6 are observed. Additionally, titration with KCl indicates that position 632 must have apositively charged residue to elicit a robust rate acceleration as a function of increasing salt. On the basisof these observations, a model for iron release from the hTF C-lobe is proposed. These studies provideinsight into the importance of charge and geometry of the amino acids at these positions as a partialexplanation for differences in behavior of individual TF family members, human serum transferrin,ovotransferrin, and lactoferrin. The studies collectively highlight important features common to both theN- and C-lobes of TF and the critical role of the receptor in iron release.
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