Characterizing the structure and function of a bacterial multi-kinase sensory complex

摘要 细菌具有惊人的能力来感知和响应细胞内和细胞外的波动 以维持细胞的动态平衡。在细菌中，环境适应通常是 由双组分系统(TCS)介导，TCS由一个组氨酸激酶(HK)传感器组成，它能磷酸化一种 响应信号检测的同源响应调节器(RR)。一旦磷酸化，RR就可以与DNA结合 并改变基因表达以促进环境适应。从历史上看，人们一直认为经典的TC 以高度线性的方式发出信号，与其他信号通路的相互作用或交叉调节最小。一个 来自我们团队和其他人的越来越多的数据提供了证据，证明一类不寻常的组氨酸激酶， 被称为HWE激酶，可以形成多蛋白质信号复合体，创造了细菌信号的新范式 转导。这些信号系统可以将来自众多环境输入的信息集成到 协调一系列生理反应。在新月形杆菌中，有一个这样的信号复合体，据此 被称为α蛋白细菌信号小体，已被发现协调调节细胞表面 附着，生物膜形成的关键初始步骤。我们已经证明了阿尔法蛋白细菌信号小体 包括a)作为分子中心蛋白的HWE激酶Skah，b)HWE激酶LovK，以及c) 经典的香港，SPDS。就个体而言，LovK和SPD在调节一般应激反应中起着关键作用 和稳定期适应。有趣的是，来自LOVK和SPD的感官信息可以被整合 通过信号体通过下游转录因子RtrA和RtrA调节细胞黏附 RtrB和假设的蛋白质RtrC。初步数据提供了信号小体包括 额外的HWE和经典的HK激酶，表明感觉复合体可以整合更广泛的范围 比之前怀疑的更多的信号。这里提出的研究采取了多学科的方法来 描述HWE信号体的结构和功能。第一个目标将使用生化方法。 和质谱学来确定Skah的分子伙伴并剖析 信号体。第二个目的是通过使用生物化学来补充信号体的结构分析 分析通过信号体组件的信号流的方法。初步证据表明 假设的蛋白质RtrC是一种隐蔽的转录因子，它的功能是HWE的关键输出 信号体。在第三个目标中，我将用X射线结晶学和X射线衍射法表征RtrC的结构和功能。 荧光记者。此外，我将使用基于FRET的生物传感器和运动分析来检查 RtrC和c-di-GMP信号之间的调节联系。HWE信号体是一个主要的模型系统 用于研究多激酶感觉系统如何按顺序检测和处理复杂的环境信息 来调节生理反应。此外，由于HWE激酶存在于许多细菌病原体中， 从这项工作中获得的见解将有助于开发针对TCS的抗菌疗法。

Abstract Bacteria have an incredible capacity to sense and respond to intra- and extracellular fluctuations in the environment in order to maintain cellular homeostasis. In bacteria, environmental adaptation is commonly mediated by two-component systems (TCS) that consist of a sensor histidine kinase (HK) that phosphorylates a cognate response regulator (RR) in response to signal detection. Upon phosphorylation, the RR can bind to DNA and alter gene expression to facilitate environmental adaptation. Classical TCS have historically been thought to signal in a highly linear manner with minimal interaction or cross-regulation with other signaling pathways. A growing body of data from our group and others provide evidence that an unusual class of histidine kinases, known as HWE kinases, can form multi-protein signaling complexes, creating a new paradigm in bacterial signal transduction. These signaling systems can integrate information from numerous environmental inputs to coordinate an array of physiological responses. In Caulobacter crescentus, one such signaling complex, hereby referred to as the Alphaproteobacterial signalosome, has been identified to coordinately regulate cellular surface attachment, a critical initial step in biofilm formation. We have shown that the Alphaproteobacterial signalosome consists of a) the HWE kinase SkaH that functions as a molecular hub protein, b) the HWE kinase LovK, and c) the classical HK, SpdS. Individually, LovK and SpdS play critical roles in modulating the general stress response and stationary phase adaptation. Interestingly, sensory information from LovK and SpdS can be integrated through the signalosome to modulate cellular adhesion through the downstream transcription factors, RtrA and RtrB, and the hypothetical protein, RtrC. Preliminary data provides evidence that the signalosome is comprised of additional HWE and classical HK kinases, suggesting that the sensory complex can integrate a broader range of signals than previously suspected. The research proposed here takes a multidisciplinary approach to characterize the structure and function of the HWE signalosome. The first aim will use biochemical approaches and mass spectrometry to identify molecular partners of SkaH and dissect direct interactions within the signalosome. The second aim will complement the structural analysis of the signalosome by using biochemical approaches to analyze the signal flow through the signalosome components. Preliminary evidence suggests that the hypothetical protein, RtrC, is a cryptic transcription factor that functions as a critical output for the HWE signalosome. In the third aim, I will characterize the structure and function of RtrC with X-ray crystallography and fluorescent reporters. Additionally, I will use FRET-based biosensors and motility assays to examine the regulatory link between RtrC and c-di-GMP signaling. The HWE signalosome serves as a prime model system for examining how multi-kinase sensory systems detect and process complex environmental information in order to regulate physiological responses. Additionally, as HWE kinases are present in many bacterial pathogens, insights gained from this work will aid in the development of antibacterial therapies that target TCS.