Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.
Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.
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DOI：
10.1371/journal.pone.0132598
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Koike T
中科院分区：
文献类型：
--
作者：
Kinoshita-Kikuta E;Kinoshita E;Eguchi Y;Yanagihara S;Edahiro K;Inoue Y;Taniguchi M;Yoshida M;Yamamoto K;Takahashi H;Sawasaki T;Utsumi R;Koike T
混合传感器激酶包含组氨酸激酶（HK）结构域、接收器结构域和含组氨酸的磷酸递质（HPt）结构域，其通过His-Asp-His-Asp磷酸中继将信号传递至其同源反应调节器。我们研究了重组EvgAS系统在大肠杆菌中的多步磷酸化，并通过使用Phos-tag SDS-PAGE进行体外磷酸化定量分析。与野生型EvgS相比，在EvgS的接收器结构域中由Ala替换Asp显著促进HK结构域中His的磷酸化。其他混合传感器激酶BarA和ArcB的类似Ala取代突变体显示出类似的特征。在足够的ATP存在下，突变体中HK结构域的自磷酸化以接近伪一级动力学有效地进行，直到磷酸化比率在60 min内达到大于95%的平台值，并且该值维持至180 min。然而，野生型EvgS和HPt结构域中His的Ala取代突变体均显示磷酸化比率小于25%，这些结果表明，磷酸化水平由接收域负调控。因此，接收器域在控制响应调节器的磷光继电器中起着至关重要的作用。此外，我们的体外测定证实了在其中Asp残基被Ala取代的EvgS突变体的HK结构域中存在类似的过度磷酸化反应，证实了从体外磷酸化分析提出的控制机制的有效性。
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.
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