Molecular Interactions in Fungal Multistep Phosphorelay Signaling Pathways

智力优势：双组分信号转导途径和扩展的多步骤His-Asp磷酸化信号转导途径控制细菌和真菌生物如何响应和适应环境胁迫。真核生物中发现的His-Asp磷酸化途径通常具有多个上游传感器激酶（HKs）和下游反应调节蛋白（RR），但几乎都依赖于单个中间组氨酸磷酸转移（HPt）蛋白进行磷酸化基团转移。虽然已经在细菌和真菌中发现了数千对HK-RR同源对，但对于控制特定途径特异性和防止单个生物体内串音的蛋白质-蛋白质相互作用知之甚少。模型酵母（Saccharomyces cerevisiae）的信号转导途径相对简单（1 HK， 1 HPt和2 rr），加上最近对Ypd1 HPt蛋白与Sln1受体结构域复合物的x射线晶体学研究，为研究磷接力信号系统中的分子相互作用提供了良好的基础。本研究的长期目标是了解磷酸盐从Ypd1流向下游响应调节因子Ssk1和Skn7的调控，作为环境胁迫的功能。该项目的主要目的是详细了解Ypd1与Sln1、Ssk1和Skn7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。具体目的是为了验证酵母磷接力信号通路内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设。将采用多学科方法，采用结构、生化和遗传方法。特异性目标1是具有同源RR结构域的Ypd1 （HPt）蛋白复合物的结构表征。特异性目的2是确定Ypd1和/或RR结构域的位点特异性突变对蛋白质结合亲和力、磷转移和相互作用特异性的影响。特异性目的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。本研究对于信号伙伴之间的相互作用以及对信号转导保真度的影响具有广泛的意义。研究结果有望首次揭示HPt-RR相互作用的关键结构特征，以及来自生命所有三个领域的多步磷接力系统中的信号特异性。更广泛的影响：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。研究与学生训练的整合是这一建议的一个重要方面。俄克拉荷马大学和爱荷华大学的本科生暑期交换项目将提供结构、生化和体内遗传方法的跨学科培训。此外，俄克拉何马大学还将为研究生或高年级本科生开设一门以实验为基础的x射线晶体学课程。学生将有机会接触到最先进的结晶机器人仪器，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.