Mutational Analysis of C-Lobe Ligands of Human Serum Transferrin:  Insights into the Mechanism of Iron Release

Abstract

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

“…Of relevance, at pH 7.4, the TFR discriminates between diferric, the two monoferric species, and apohTF, although the basis of this discrimination has not been explained (24 -26). Significantly, our studies with authentic monoferric hTF constructs established that each lobe contributes equally (and non-additively) to the binding energy of this interaction with the TFR (15). Clearly a structure of apo-hTF is required to determine whether a change in orientation of the two lobes could provide both a rationale for discrimination and further insight into the receptor interaction.…”

Differential regulation of transferrin 1 and 2 in Aedes aegypti

“…He et al (1997) mutated Y188 to F (a non-coordinating ligand) in HsTf and abolished iron binding in the N lobe. Likewise a mutation of the corollary residue Y517 to F also abolished iron binding in the C lobe (Mason et al, 2005). Although the effects of mutations Y517N or H585N on iron-binding activity have not been studied, iron-binding activity, as well as release, could be compromised by these substitutions.…”

A loop in the N‐lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping

“…Our previous work (Mason et al ., 1997) provided compelling evidence that the N -lobe in full-length hTF plays a role in the high affinity binding of Fe2 hTF to the TFR. Further proof came from equilibrium binding experiments showing that the free energy of binding of each monoferric hTF was equivalent and, significantly, was not additive (Mason et al ., 2005). The current work now provides additional irrefutable evidence that both lobes are required for highest affinity binding and that the loop region of the N -lobe contributes significantly to this binding.…”