Molecular Interactions in Fungal Multistep Phosphorelay Signaling Pathways

真菌多步磷酸中继信号通路中的分子相互作用

• 批准号: 1158319
• 负责人: Ann West
• 金额: $ 62.65万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158319&HistoricalAwards=false
• 关键词: Molecular; Interactions; Fungal; Multistep; Phosphorelay

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.

智力优势：双组分信号转导途径和扩展的多步His-Asp磷酸化信号转导途径控制细菌和真菌生物体如何响应和适应环境胁迫。 在真核生物中发现的His-Asp磷酸转移途径通常具有多个上游传感激酶（HK）和下游反应调节（RR）蛋白，但几乎所有都依赖于单个中间组氨酸磷酸转移（HPt）蛋白进行磷酰基转移。 尽管在细菌和真菌中已经鉴定出数千个HK-RR同源对，但对蛋白质-蛋白质相互作用的了解很少，这些蛋白质-蛋白质相互作用控制特定途径内的特异性并防止单个生物体内的串扰。 相对简单的信号转导途径（一个HK，一个HPt和两个RR）在模型酵母酿酒酵母，连同最近的X-射线晶体学研究的Ypd 1 HPt蛋白与Sln 1接收器结构域的复杂性，提供了一个很好的基础内的phosphorelay信号系统的分子相互作用的调查。 这项研究的长期目标是了解磷酸盐从Ypd 1流向下游响应调节因子Ssk 1和Skn 7的调节，作为环境压力的函数。 本项目的主要目标是详细了解Ypd 1与Sln 1相关的三个同源反应调节结构域相互作用的结构、生化和功能意义，Ssk 1和Skn 7.具体的目的是为了验证酵母磷酸化信号通路中的分子相互作用受到外部环境信号和磷酸化状态的影响的假设。相互作用的信号伙伴。 将采用结构、生物化学和遗传学方法的多学科方法。 具体目标1是具有同源RR结构域的Ypd 1（HPt）蛋白复合物的结构表征。 具体目标2是确定Ypd 1和/或RR结构域中的位点特异性突变对蛋白结合亲和力、磷酸转移和相互作用特异性的影响。 具体目标3是确定在SLN 1通路中影响Ypd 1-RR相互作用的突变的体内后果。拟议的研究对于信号伴侣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生命所有三个领域的多步磷酸化中继系统中有助于HPt-RR相互作用和信号特异性的关键结构特征。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，具体而言，蛋白质磷酸化的作用及其对调节蛋白质相互作用的影响。 研究和学生培养的一体化是这一建议的一个重要方面。 俄克拉荷马州大学和爱荷华州大学的本科生暑期交流项目将提供结构、生物化学和体内遗传方法方面的跨学科培训。 此外，将在俄克拉荷马州大学的研究生水平或高年级本科生顶点水平开发和提供基于实验室的动手操作的X射线晶体学课程。 学生将有机会获得最先进的（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。