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

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 tra nsmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-bind ing protein and the CitA kinase domain (amino acids 327-547) showed constit utive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of cite was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphoryl ation 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 (C itAP(His)). Purified CitAP(His) bound citrate, but none of the other tri- a nd dicarboxylates tested, with high affinity (K-D approximate to 5 mu M at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (Delta H = -76.3 kJ mol(-1)), whereas the entropy change was opposed (-T Delta S = +46.3 kJ mo l(-1)). The pH dependency of the binding reaction indicated that the dianio nic form H-citrate(2-) is the citrate species recognized by CitAP(His). In the presence of Mg2+ ions, the dissociation constant increased significantl y, suggesting that the Mg-citrate complex is not bound by CitAP(His) This w ork defines the periplasmic domain of CitA as a highly specific citrate rec eptor and elucidates the binding characteristics of CitAP(His).