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Functional characterisation of two ferric-ion coordination modes of TtFbpA, the periplasmic subunit of an ABC-type iron transporter from Thermus thermophilus HB8†
a
Yoshitaka
Moriwaki,
b
Ken
Okamoto,
a
Tohru
Terada,
cd
Kentaro
Shimizu,
bd
Takehiro
Kamiya,
a
Toru
Fujiwara,
a
Tomiko
Asakura,
a
Michio
Suzuki,
a
Koji
Nagata
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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.
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