Protein-protein interactions that regulate the energy stress activation of σB in Bacillus subtilis
Protein-protein interactions that regulate the energy stress activation of σB in Bacillus subtilis
复制标题
DOI：
10.1128/jb.184.20.5583-5589.2002
复制
发表时间：
2002-10-01
影响因子：
3.2
通讯作者：
Yudkin, MD
中科院分区：
文献类型：
--
作者：
Delumeau, O;Lewis, RJ;Yudkin, MD
σ（B）是控制枯草芽孢杆菌中一般应激反应的替代物或因子。在没有应激的情况下，σ（B）受反σ因子RsbW负调节。RsbW也是一种可以磷酸化RsbV的蛋白激酶。当细胞受到应激时，RsbW与未磷酸化的RsbV结合，所述未磷酸化的RsbV由应激激活的两种磷酸酶（RsbU和RsbP）从RsbV的磷酸化形式产生。我们现在报告RsbW的ATP的Km值和ADP的Ki值（分别为0.9和0.19 mM），这加强了RsbW的激酶活性在体内由这些核苷酸的比例直接调节的想法。纯化为二聚体的RsbW与RsbV和sigma（B）形成具有不同化学计量的复合物，即，RsbW（2）-RsbV（2）和RsbW（2）-sigma（1）（B）。如通过表面等离子体共振测定的，发现RsbW-RsbV和RsbW-sigma（B）相互作用的解离常数相似（分别为63和92 nM）。尽管如此，在竞争试验中通过非变性聚丙烯酰胺凝胶电泳对复合物的分析表明，RsbW对RsbV的亲和力远高于对sigma（B）的亲和力。应激前测定的RsbV、RsbW（作为单体）和sigma（B）的细胞内浓度相似（分别为1.5、2.6和0.9 μ M）。乙醇胁迫后，它们都增加了。RsbV的增加最大，其浓度达到13 μ M，而RsbW（作为单体）和sigma（B）的浓度分别达到11.8和4.9 μ M。我们得出结论，RsbW对RsbV的亲和力高于对sigma（B）的亲和力，而不是RsbV和sigma（B）浓度的差异，是RsbW转换为未磷酸化RsbV的驱动力。
sigma(B) is an alternative or factor that controls the general stress response in Bacillus subtilis. In the absence of stress, sigma(B) is negatively regulated by anti-sigma factor RsbW. RsbW is also a protein kinase which can phosphorylate RsbV. When cells are stressed, RsbW binds to unphosphorylated RsbV, produced from the phosphorylated form of RsbV by two phosphatases (RsbU and RsbP) which are activated by stress. We now report the values of the K-m for ATP and the K-i for ADP of RsbW (0.9 and 0.19 mM, respectively), which reinforce the idea that the kinase activity of RsbW is directly regulated in vivo by the ratio of these nucleotides. RsbW, purified as a dimer, forms complexes with RsbV and sigma(B) with different stoichiometries, i.e., RsbW(2)-RsbV(2) and RsbW(2)-sigma(1)(B). As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-sigma(B) interactions were found to be similar (63 and 92 nM, respectively). Nonetheless, an analysis of the complexes by nondenaturing polyacrylamide gel electrophoresis in competition assays suggested that the affinity of RsbW, for RsbV is much higher than that for sigma(B). The intracellular concentrations of RsbV, RsbW (as a monomer), and sigma(B) measured before stress were similar (1.5, 2.6, and 0.9 muM, respectively). After ethanol stress they all increased. The increase was greatest for RsbV, whose concentration reached 13 muM, while those of RsbW (as a monomer) and sigma(B) reached 11.8 and 4.9 muM, respectively. We conclude that the higher affinity of RsbW for RsbV than for sigma(B), rather than a difference in the concentrations of RsbV and sigma(B), is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.