Molecular Interactions in Fungal Multistep Phosphorelay Signaling Pathways

智力优势：双组分信号转导通路和扩展的多步His-Asp磷酸转导通路控制细菌和真菌生物对环境压力的反应和适应。在真核生物中发现的His-Asp磷酸传递途径通常具有多个上游感受器蛋白(HKS)和下游反应调节蛋白(RR)，但几乎所有的蛋白都依赖于单一的中间组氨酸磷酸转移(HPT)蛋白进行磷酸基转移。尽管已在细菌和真菌中鉴定出数千对HK-RR同源对，但对控制特定途径内的特异性并防止单个有机体内的串扰的蛋白质-蛋白质相互作用知之甚少。酿酒酵母模型中信号转导途径的相对简单(一个HK，一个HPT和两个RRs)，加上最近对Ypd1 HPT蛋白与SLn1受体结构域复合体的X射线结晶学研究，为研究磷传递信号系统中的分子相互作用提供了良好的基础。这项研究的长期目标是了解从Ypd1到下游反应调节因子Ssk1和SKn7的磷酸盐流动的调节，作为环境胁迫的函数。本项目的主要目的是详细了解Ypd1与SLn1、Ssk1和Skn7三个同源反应调节结构域的相互作用的结构、生化和功能意义。具体目的是检验酵母磷传递信号通路中的分子相互作用受外部环境信号和相互作用信号伙伴的磷酸化状态影响的假说。将采取使用结构、生物化学和遗传方法的多学科方法。具体目标1是具有同源RR结构域的Ypd1(HPT)蛋白复合体的结构特征。具体目的2是确定Ypd1和/或RR结构域的定点突变对蛋白结合亲和力、磷转移和相互作用的特异性的影响。具体目的3是确定影响SLN1通路中Ypd1-RR相互作用的突变在体内的后果。所提出的研究对于信号伙伴如何相互作用并影响信号转导的保真度具有广泛的意义。这些结果有望首次揭示影响HPT-RR相互作用的关键结构特征和生活中所有三个领域的多步磷酸传递系统中的信号特异性。广泛影响：该项目的结果有望为控制信号转导途径的原理提供重要的新见解，特别是蛋白质磷酸化的作用及其对调节蛋白质-蛋白质相互作用的影响。研究和学生培训的结合是这项提议的一个重要方面。俄克拉荷马大学和爱荷华大学的本科生暑期交流项目将提供结构、生化和体内遗传方法的跨学科培训。此外，俄克拉荷马大学还将开发一门以实验室为基础的实践X射线结晶学课程，并在研究生水平或高级本科生水平上提供这一课程。学生将有机会接触到最先进的(NSF资助的)结晶机器人仪器，并应用X射线衍射技术来解决生物大分子的三维结构。

Intellectual Merit: Two-component signal transduction pathways and expanded multi-step His-Asp phosphorelay signaling pathways control how bacteria and fungal organisms respond and adapt to environmental stress. The His-Asp phosphorelay pathways found in eukaryotes frequently feature multiple upstream sensor kinases (HKs) and downstream response regulator (RR) proteins, yet nearly all depend on a single intermediate histidine phosphotransfer (HPt) protein for phosphoryl group transfer. Although several thousands of HK-RR cognate pairs have been identified in bacteria and fungi, very little is understood about protein-protein interactions that govern specificity within a particular pathway and prevent cross-talk within a single organism. The relative simplicity of the signal transduction pathway (one HK, one HPt and two RRs) in the model yeast Saccharomyces cerevisiae, together with recent X-ray crystallographic studies of the Ypd1 HPt protein in complex with the Sln1 receiver domain, provides an excellent foundation for the investigation of molecular interactions within a phosphorelay signaling system. The long-term goal of this research is to understand regulation of phosphate flow from Ypd1 to the downstream response regulators, Ssk1 and Skn7, as a function of environmental stress. The main objective of this project is to achieve a detailed understanding of the structural, biochemical and functional implications of Ypd1 interactions with the three homologous response regulator domains associated with Sln1, Ssk1 and Skn7.The specific aims are designed to test the hypothesis that molecular interactions within the yeast phosphorelay signaling pathway are influenced by external environmental signals and the phosphorylation state of the interacting signaling partners. A multidisciplinary approach using structural, biochemical and genetic approaches will be taken. Specific Aim 1 is structural characterization of Ypd1 (HPt) protein complexes with cognate RR domains. Specific Aim 2 is to determine the effect of site-specific mutations in Ypd1 and/or RR domains on protein binding affinity, phosphotransfer and specificity of interaction. Specific Aim 3 is to determine the in vivo consequences of mutations that affect Ypd1-RR interactions in the SLN1 pathway.The proposed research has broad significance with respect to how signaling partners interact with each other and influence fidelity of signal transduction. The results are expected to reveal, for the first time, key structural features that contribute to HPt-RR interactions and signaling specificity within multistep phosphorelay systems from all three domains of life.Broader Impacts: Results from this project are expected to provide significant new insight into the principles that govern signal transduction pathways, specifically, the role of protein phosphorylation and its impact on regulating protein-protein interactions. The integration of research and student training is an important aspect of this proposal. A summer exchange program for undergraduate students at the University of Oklahoma and the University of Iowa will provide cross-disciplinary training in structural, biochemical and in vivo genetic approaches. In addition, a hands-on laboratory-based X-ray crystallography course will be developed and offered at the graduate-level or senior undergraduate capstone level at University of Oklahoma. Students will have the opportunity to access state-of-the-art (NSF-funded) crystallization robotics instrumentation and apply X-ray diffraction techniques to solve the three-dimensional structure of biomacromolecules.