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PLoS One. 2015 Jul 7;10(7):e0132598. doi: 10.1371/journal.pone.0132598. eCollection 2015.

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

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Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Japan.
Department of Frontier Bioscience, Hosei University, Koganei, Japan.
Division of Cell-Free Sciences, Proteo-Science Center, Ehime University, Matsuyama, Japan.
Department of Bioscience, Graduate School of Agriculture, Kinki University, Nara Japan.


Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].

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