Mechanism of metal ion-induced activation of a two-component sensor kinase
Mechanism of metal ion-induced activation of a two-component sensor kinase
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DOI：
10.1042/bcj20180577
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发表时间：
2019-01-15
影响因子：
4.1
通讯作者：
McEvoy, Megan M.
中科院分区：
文献类型：
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作者：
Affandi, Trisiani;McEvoy, Megan M.
双组分系统（TCSs）对于细菌感知、响应并适应不断变化的环境至关重要，例如周质中Cu（I）/Ag（I）离子浓度的升高。在大肠杆菌中，CusS - CusR双组分系统在周质中Cu（I）/Ag（I）浓度升高时上调cusCFBA基因，以帮助维持金属离子稳态。CusS组氨酸激酶是一种同二聚体整合膜蛋白，它与周质中的Cu（I）/Ag（I）结合，并将信号传递至其胞质激酶结构域。然而，周质中的金属结合如何激活胞质中的自磷酸化机制尚不清楚。在此，我们报道CusS中两个金属离子结合位点中只有一个能增强传感器结构域的二聚化。利用纳米圆盘技术研究全长CusS，我们发现传感器结构域中金属诱导的二聚化会触发胞质结构域中的激酶活性。我们还研究了CusS胞质结构域中的自磷酸化以及CusS和CusR之间的磷酸转移。体外分析表明，CusS在组氨酸咪唑的N1位置将其保守的H271残基自磷酸化。磷酸基团在一个需要第51位保守天冬氨酸的反应中被应答调节因子CusR去除。对自磷酸化或磷酸受体残基发生突变的CusS和CusR变体进行体内功能分析表明，磷酸转移事件对于大肠杆菌的金属抗性至关重要。生化分析表明，CusS二聚体利用顺式机制进行自磷酸化。我们的结果支持一种信号转导模型，即胞质结构域中的旋转和弯曲运动维持自磷酸化模式。
Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS-CusR TCS up-regulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu (I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion-binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. In vitro analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses in vivo of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in E. coli. Biochemical analysis shows that the CusS dimer autophosphorylates using a cis mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation.