Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator.
EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein-labelled OmpR (Fl-OmpR) with EnvZc was determined to be 1.96 +/- 0.28 micro M. We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl-OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 +/- 0.25 micro M and 0.89 +/- 0.14 micro M, respectively, under the conditions used. The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl-OmpR or Fl-OmpR-P as well as with OmpR or OmpR-P as a substrate. We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.
Yoshida
Takeshi
T
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Cai
Sheng jian
Sj
Inouye
Masayori
M
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000502:physiology; D029968:Escherichia coli Proteins; D019793:Fluorescein / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase; D009097:Multienzyme Complexes / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015534:Trans-Activators / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases; D019793:Fluorescein