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 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 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 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 approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 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.
Kaspar
S
S
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Perozzo
R
R
Reinelt
S
S
Meyer
M
M
Pfister
K
K
Scapozza
L
L
Bott
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D019343:Citric Acid / Q000378:metabolism; D003001:Cloning, Molecular; D000071677:Histidine Kinase; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D019897:Periplasm / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; C107075:CitB protein, Klebsiella pneumoniae; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019343:Citric Acid; D011494:Protein Kinases; D000071677:Histidine Kinase