The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor

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Abstract
The two‐component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane‐bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE–CitAC) composed of the maltose‐binding protein and the CitA kinase domain (amino acids 327–547) showed constitutive autokinase activity and transferred the γ‐phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H‐350 and D‐56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB–D56N formed a stable complex with MalE–CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45–176) was overproduced as a soluble, cytoplasmic protein with a C‐terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri‐ and dicarboxylates tested, with high affinity (KD ≈ 5 μM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (ΔH = −76.3 kJ mol−1), whereas the entropy change was opposed (−TΔS = + 46.3 kJ mol−1). The pH dependency of the binding reaction indicated that the dianionic form H‐citrate2− is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg–citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.