Structural and functional analysis of a dynamic ABA signaling complex

描述（由申请人提供）：大多数信号传导途径涉及快速解离的不稳定的动态蛋白质复合物，因此难以通过高分辨率结构研究进行分析。在这个提议中，我们将使用蛋白质工程来确定关键的植物胁迫激素脱落酸（阿坝）的动态信号复合物的晶体结构。阿坝是一种古老的信号分子，存在于植物、真菌和从海绵到人类的后生动物中。在植物中，阿坝是一种必需的激素，是保护植物免受干旱、寒冷和盐等非生物胁迫的中心调节因子。这些压力是作物生产的主要限制因素，因此是造成粮食短缺造成营养不良的主要因素。这与人类健康有关，因为营养不良导致了全球50%以上的人类疾病，包括癌症和传染病。了解阿坝信号转导的详细机制，将是至关重要的阿坝途径在植物中的基因工程提供的机制基础。在阿坝信号传导的中心是AMPK相关蛋白激酶家族，其通过磷酸化转录因子、离子通道和第二信使产生酶来传递阿坝信号。这些激酶受2C型蛋白磷酸酶（PP 2Cs）和细胞内阿坝受体的控制。在该提案中，证据是存在的四元信号复合物，包含受体，阿坝，PP 2Cs，和SnRK 2。我们将使用蛋白质工程来稳定这些复合物，并使它们适合X射线晶体学。这些复合物的结构将提供重要的洞察这些复合物的功能，并将确定所有蛋白质-蛋白质和蛋白质-ABA相互作用的背景下的复杂的关键相互作用残基。我们将对这些残基进行突变，以确定这些相互作用在生物化学和基于细胞的测定中以及在转基因植物体内的功能。该项目的成果将为阿坝信号传导中受体、阿坝、PP 2C和SnRK 2相互作用的结构理解提供一个全面的框架，从而为调节植物中的阿坝途径以提高其水分利用效率和粮食产量提供机制基础。 公共卫生相关性：仅因食物短缺导致的营养不良就造成了全球50%以上的人类疾病，包括癌症和传染病。粮食生产的主要限制是全球淡水资源的稀缺，目前超过70%的淡水用于农业。提高作物水分利用效率是解决这一关键问题的途径之一，但这一途径的一个关键障碍是我们对植物响应水分胁迫的分子机制认识不足，本项目从研究植物科普水分胁迫的核心调节因子脱落酸（阿坝）的信号传导机制入手，着手解决这一关键问题。

DESCRIPTION (provided by applicant): Most signaling pathways involve labile, dynamic protein complexes that rapidly dissociate and that are therefore notoriously difficult to analyze by high resolution structural studies. In this proposal we will use protein engineering to determine the crystal structure of a dynamic signaling complex of the crucial plant stress hormone abscisic acid (ABA). ABA is an ancient signaling molecule that is found in plants, fungi, and metazoans ranging from sponges to humans. In plants, ABA is an essential hormone and the central regulator to protect plants against abiotic stresses such as drought, cold, and salinity. These stresses are major limiting factors in crop production and therefore main contributors to malnutrition due to food shortage. This is relevant to human health because malnutrition contributes to more than 50% of human disease worldwide, including cancer and infectious diseases. Understanding the detailed mechanism of ABA signaling will be critical to provide a mechanistic basis for genetic engineering of ABA pathways in plants. At the center of ABA signaling are a family of AMPK-related protein kinases that relay the ABA signal by phosphorylating transcription factors, ion channels, and second-messenger-generating enzymes. These kinases are under the control of type 2C protein phosphatases (PP2Cs) and intracellular ABA receptors. In this proposal evidence is presented for the existence of quaternary signaling complexes that contain the receptors, ABA, PP2Cs, and SnRK2s. We will use protein engineering to stabilize these complexes and make them amenable to X-ray crystallography. The structure of these complexes will provide important insight into the function of these complexes and will identify the key interacting residues for all protein-protein and protein-ABA interactions in the context of the complex. We will mutate these residues to determine the function of these interactions in biochemical and cell-based assays as well as in vivo in transgenic plants. The outcome of this project will provide a comprehensive framework for structural understanding of receptor, ABA, PP2C, and SnRK2 interactions in ABA signaling and will thus provide a mechanistic basis for modulating ABA pathways in plants to improve their water use efficiency and food production. PUBLIC HEALTH RELEVANCE: Malnutrition due to food shortage alone contributes to more than 50% of human disease worldwide, including cancer and infectious diseases. The major limitation for food production is the scarceness of fresh water resources at the global scale where >70% of fresh water is currently used by agriculture. One solution to this critical problem is to increase the water use efficiency of crop plants, but a critical barrier toward this solutionis our poor understanding of molecular mechanisms underlying plant responses to water stress; this project begins to address this critical problem by studying the signaling mechanism of abscisic acid (ABA), which is the central regulator in plants to cope with water stress.