Cargando…
Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases
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 Ev...
Autores principales: | , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494823/ https://www.ncbi.nlm.nih.gov/pubmed/26151934 http://dx.doi.org/10.1371/journal.pone.0132598 |
_version_ | 1782380158496276480 |
---|---|
author | Kinoshita-Kikuta, Emiko Kinoshita, Eiji Eguchi, Yoko Yanagihara, Shiho Edahiro, Keisuke Inoue, Yuki Taniguchi, Momoka Yoshida, Myu Yamamoto, Kaneyoshi Takahashi, Hirotaka Sawasaki, Tatsuya Utsumi, Ryutaro Koike, Tohru |
author_facet | Kinoshita-Kikuta, Emiko Kinoshita, Eiji Eguchi, Yoko Yanagihara, Shiho Edahiro, Keisuke Inoue, Yuki Taniguchi, Momoka Yoshida, Myu Yamamoto, Kaneyoshi Takahashi, Hirotaka Sawasaki, Tatsuya Utsumi, Ryutaro Koike, Tohru |
author_sort | Kinoshita-Kikuta, Emiko |
collection | PubMed |
description | 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. The receiver domain therefore plays a crucial role in controlling the phosphorelay to the response regulator. Furthermore, our in vitro 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. |
format | Online Article Text |
id | pubmed-4494823 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-44948232015-07-15 Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases Kinoshita-Kikuta, Emiko Kinoshita, Eiji Eguchi, Yoko Yanagihara, Shiho Edahiro, Keisuke Inoue, Yuki Taniguchi, Momoka Yoshida, Myu Yamamoto, Kaneyoshi Takahashi, Hirotaka Sawasaki, Tatsuya Utsumi, Ryutaro Koike, Tohru PLoS One Research Article 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. The receiver domain therefore plays a crucial role in controlling the phosphorelay to the response regulator. Furthermore, our in vitro 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. Public Library of Science 2015-07-07 /pmc/articles/PMC4494823/ /pubmed/26151934 http://dx.doi.org/10.1371/journal.pone.0132598 Text en © 2015 Kinoshita-Kikuta et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kinoshita-Kikuta, Emiko Kinoshita, Eiji Eguchi, Yoko Yanagihara, Shiho Edahiro, Keisuke Inoue, Yuki Taniguchi, Momoka Yoshida, Myu Yamamoto, Kaneyoshi Takahashi, Hirotaka Sawasaki, Tatsuya Utsumi, Ryutaro Koike, Tohru Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title | Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title_full | Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title_fullStr | Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title_full_unstemmed | Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title_short | Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases |
title_sort | functional characterization of the receiver domain for phosphorelay control in hybrid sensor kinases |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494823/ https://www.ncbi.nlm.nih.gov/pubmed/26151934 http://dx.doi.org/10.1371/journal.pone.0132598 |
work_keys_str_mv | AT kinoshitakikutaemiko functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT kinoshitaeiji functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT eguchiyoko functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT yanagiharashiho functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT edahirokeisuke functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT inoueyuki functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT taniguchimomoka functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT yoshidamyu functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT yamamotokaneyoshi functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT takahashihirotaka functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT sawasakitatsuya functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT utsumiryutaro functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases AT koiketohru functionalcharacterizationofthereceiverdomainforphosphorelaycontrolinhybridsensorkinases |