Signal integration and transmission by the Cpx-TCS of Gram-negative bacteria

细菌具有双组分系统（TCS）来应对环境变化。然而，TCS信号转导的机制细节仍然知之甚少。革兰氏阴性菌的Cpx-TCS是目前研究最多的TCS之一，由感知激酶CpxA、应答调节蛋白CpxR和辅助蛋白CpxP组成。它对各种信号（如pH值、渗透压或错误折叠的蛋白质）诱导的包膜应力作出反应。我们已经确定，重组的传感器激酶CpxA的所有三种催化活性都可以通过不同的信号进行特异性调节：(i) CpxP抑制自磷酸化，（ii）周围质麦芽糖结合蛋白的折叠缺陷变体刺激CpxR的转磷酸化，以及（iii）脂质调节磷酸化CpxR的去磷酸化。此外，我们对CpxP的结构和功能研究首次揭示了CpxP如何与CpxA相互作用，并作为错误折叠的菌毛亚基的传感器。我建议继续我们对Cpx-TCS信号整合的研究，特别强调CpxP的功能：(I)抑制CpxA， （ii）菌毛识别和（iii）与外质atp非依赖性蛋白酶DegP的功能相互作用。此外，利用我们对Cpx-TCS信号整合的现有知识，我们将研究生物学中的一个关键问题：信号是如何穿过膜的？为此，我们开发了“Membrane-SPINE”，这是一种验证蛋白质-蛋白质相互作用的方法，并创建了CpxA与CpxR复合物的催化核心的结构模型。我们将解决相互作用的关键残基，并在CpxA的催化活性特定调节后，通过同源双功能交联和EPR确定它们在信号传导过程中的相对重定向。这两门课程将有助于对信号集成和信号传输的总体理解。

Bacteria are equipped with two-component systems (TCS) to cope with environmental changes. However, the mechanistic details in TCS signal transduction are still poorly understood. The Cpx-TCS of Gram-negative bacteria is one of the best studied TCS and is composed of the sensor kinase CpxA, the response regulator CpxR, and the auxiliary protein CpxP. It responds to envelope stress which is induced by various signals e.g. pH, osmolarity or misfolded proteins. We have established that all three catalytic activities of the reconstituted sensor kinase CpxA can be modulated specifically by different signals: (i) CpxP inhibits autophosphorylation, (ii) a folding-deficient variant of periplasmic maltose binding protein stimulates transphosphorylation to CpxR, and (iii) lipids modulate dephosphorylation of phosphorylated CpxR. Moreover, our structural and functional studies on CpxP provide first insight how CpxP interacts with CpxA and serves as sensor for misfolded pilus subunits. I propose to continue our investigations on signal integration by the Cpx-TCS with special emphasis on the functions of CpxP: (i) inhibition of CpxA, (ii) pilus recognition and (iii) functional interaction with the periplasmic ATP-independent protease DegP. In addition and by taking advantage of our existing knowledge on signal integration by the Cpx-TCS, we will study one of the key questions in biology: How does a signal transverse a membrane? To this end, we have developed ‘Membrane-SPINE’, a method that will allow us to verify protein-protein interactions and have created a structural model of the catalytic core of CpxA in complex with CpxR. We will address critical residues for interaction and determine their relative reorientation during signalling by homobifunctional cross-linking and EPR after specific modulation of the catalytic activities of CpxA. Both subjects will contribute to the general understanding of signal integration and signal transmission.