Perraud A L, Kimmel B, Weiss V, Gross R
Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut, Biozentrum der Universität Würzburg, Germany.
Mol Microbiol. 1998 Mar;27(5):875-87. doi: 10.1046/j.1365-2958.1998.00716.x.
Despite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-component systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.
尽管存在高度保守的信号传导模块，但不同双组分系统之间显著的交叉通讯却很少被观察到。结构域交换以及对游离信号传导模块的表征，使我们能够在体外表征介导特异性并负责非正统Bvg和Evg双组分系统磷传递高保真度的蛋白质结构域。在等摩尔条件下，纯化的BvgA和EvgA蛋白的显著体外磷酸化仅分别由它们的组氨酸激酶BvgS和EvgS实现。一种由BvgS传递结构域和HPt结构域以及EvgS接收结构域组成的杂合组氨酸激酶（BvgS-TO-EvgS-R）能够磷酸化BvgA，但不能磷酸化EvgA。相反，由BvgS传递结构域以及EvgS接收和HPt结构域组成的杂合蛋白（BvgS-T-EvgS-RO）不能磷酸化BvgA，但能有效地磷酸化EvgA。这些结果表明，传感蛋白的C末端HPt结构域赋予非正统双组分系统对相应调节蛋白的高特异性。就响应调节蛋白而言，接收结构域而非输出结构域有助于与组氨酸激酶的特异性相互作用，因为由EvgA接收结构域和BvgA输出结构域组成的杂合蛋白只能被EvgS蛋白磷酸化。
