Functional characterisation of two ferric-ion coordination modes of TtFbpA, the periplasmic subunit of an ABC-type iron transporter from Thermus thermophilus HB8

Abstract

The ferric ion binding protein A of Thermus thermophilus HB8 (TtFbpA) is the periplasmic subunit of an ABC-type iron transporter. Two Fe³⁺-bound crystal structures at pH 5.5 and pH 7.5 and one apo structure have been reported for TtFbpA. In addition to three Tyr residues, TtFbpA coordinates with Fe³⁺ using two monodentate HCO₃⁻ and one H₂O to form a six-coordinated mode at pH 5.5 or one bidentate CO₃²⁻ to form a five-coordinated mode at pH 7.5. We investigated the biological significance of these Fe³⁺-bound forms of TtFbpA and the synergistic anions (HCO₃⁻ and CO₃²⁻). Quantum mechanical calculations in silico indicated that only these coordination modes were plausible out of six possibilities. Comparison of the crystal structures revealed a key motif, RZX₁X₂L(I/V), that would couple the Fe³⁺ coordination mode and the TtFbpA protein conformation. Both gel filtration chromatography and isothermal titration calorimetry showed that TtFbpA could bind Fe³⁺ at pH 7.5 but not at pH 5.5. Isothermal titration calorimetry also revealed that the binding at pH 7.5 was a three-step endothermic reaction that required NaHCO₃. These results indicate that the holo structure at pH 5.5 is unstable in solution and may correspond to a transition state of TtFbpA–Fe³⁺ binding at pH 7.5 because HCO₃⁻ is much more abundant than CO₃²⁻ at both pH values. Reorganisation of the synergistic ions and coupled protein conformational change will occur to form the stable TtFbpA–Fe³⁺ complex at pH 7.5, but not at pH 5.5. Identification of such a transition state will contribute to molecular design of novel FbpA inhibitors.