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 (CitAP(His)). Purified CitAP(His), bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (K(D) ≃ 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 CitAP(His). In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAP(His). This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAP(His).

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Kaspar, S., Perozzo, R., Reinelt, S., Meyer, M., Pfister, K., Scapozza, L., & Bott, M. (1999). The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor. Molecular Microbiology, 33(4), 858–872. https://doi.org/10.1046/j.1365-2958.1999.01536.x

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