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.

中文翻译：

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TtFbpA的两个铁离子配位模式的功能表征，TtFbpA是嗜热栖热菌HB8的ABC型铁转运蛋白的周质亚基。

嗜热栖热菌HB8（TtFbpA）的铁离子结合蛋白A是ABC型铁转运蛋白的周质亚基。对于TtFbpA，已经报道了在pH 5.5和pH 7.5时有两个Fe3 +结合的晶体结构和一个载脂蛋白结构。除三个Tyr残基外，TtFbpA与Fe3 +配合使用两个单齿HCO3-和一个H2O在pH 5.5时形成六配位模式，或在pH 7.5时形成二齿CO32-形成五配位模式。我们研究了TtFbpA与这些Fe3 +结合形式和协同阴离子（HCO3-和CO32-）的生物学意义。计算机进行的量子力学计算表明，在六种可能性中，只有这些协调模式才是合理的。晶体结构的比较揭示了一个关键基序RZX1X2L（I / V），它将结合Fe3 +配位模式和TtFbpA蛋白构象。凝胶过滤色谱法和等温滴定量热法均显示，TtFbpA可以在pH 7.5时结合Fe3 +，而在pH 5.5时不结合。等温滴定量热法还显示，在pH 7.5下的结合是需要NaHCO3的三步吸热反应。这些结果表明，pH 5.5时的整体结构在溶液中不稳定，并且可能对应于pH 7.5时TtFbpA-Fe3 +结合的过渡态，因为在两个pH值下HCO3-比CO32-都丰富得多。在pH 7.5而不是pH 5.5时，将发生协同离子的重组和偶联的蛋白质构象变化，以形成稳定的TtFbpA-Fe3 +复合物。鉴定这种过渡态将有助于新型FbpA抑制剂的分子设计。