Structural and functional analysis of a dynamic ABA signaling complex

动态 ABA 信号复合物的结构和功能分析

• 批准号: 8346496
• 负责人: Karsten Melcher
• 金额: $ 36.1万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8346496
• 关键词: Abscisic Acid; Address; Agriculture; Architecture; Binding; Biochemical; Biological Assay; Catalytic Domain; Cells; Communicable Diseases; Complex; Data; Droughts; Enzymes; Family; Food; Fresh Water; Generations; Genetic Engineering; Goals; Harvest; Health; Hormones; Human; Individual; Ion Channel; KRP protein; Knowledge; Ligands; Mainstreaming; Malignant Neoplasms; Malnutrition; Mediating; Molecular; Mutate; Outcome; Pathway interactions; Phosphotransferases; Physiological; Plant Physiology; Plants; Porifera; Production; Protein Engineering; Proteins; Regulation; Research; Resolution; Resources; Science; Second Messenger Systems; Seeds; Signal Pathway; Signal Transduction; Signaling Molecule; Solutions; Stress; Structure; Technology; Testing; Transcriptional Activation; Transgenic Plants; Water; Water Stress; Water consumption; X-Ray Crystallography; base; biological adaptation to stress; coping; food shortage; human disease; improved; in vivo; insight; plant fungi; protein complex; protein phosphatase 2C; protein protein interaction; receptor; receptor binding; receptor function; response; second messenger; transcription factor

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.

描述（由申请人提供）：大多数信号传导途径涉及不稳定的动态蛋白质复合物，这些蛋白质复合物会快速解离，因此很难通过高分辨率结构研究进行分析。在本提案中，我们将利用蛋白质工程来确定重要的植物应激激素脱落酸 (ABA) 的动态信号复合物的晶体结构。 ABA 是一种古老的信号分子，存在于植物、真菌和后生动物（从海绵到人类）中。在植物中，ABA 是一种重要的激素，也是保护植物免受干旱、寒冷和盐度等非生物胁迫的中央调节剂。这些压力是作物生产的主要限制因素，因此也是导致粮食短缺导致营养不良的主要原因。这与人类健康相关，因为全球 50% 以上的人类疾病，包括癌症和传染病，都是由营养不良造成的。了解ABA信号传导的详细机制对于为植物ABA途径的基因工程提供机制基础至关重要。 ABA 信号传导的核心是 AMPK 相关蛋白激酶家族，它们通过磷酸化转录因子、离子通道和第二信使生成酶来传递 ABA 信号。这些激酶受 2C 型蛋白磷酸酶 (PP2C) 和细胞内 ABA 受体的控制。该提案提供了包含受体 ABA、PP2C 和 SnRK2 的四元信号复合物存在的证据。我们将利用蛋白质工程来稳定这些复合物，并使它们适合 X 射线晶体学分析。这些复合物的结构将为了解这些复合物的功能提供重要的见解，并将识别复合物背景下所有蛋白质-蛋白质和蛋白质-ABA 相互作用的关键相互作用残基。我们将突变这些残基，以确定这些相互作用在生化和基于细胞的测定以及转基因植物体内的功能。该项目的成果将为 ABA 信号传导中受体、ABA、PP2C 和 SnRK2 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 ABA 途径以提高其水分利用效率和粮食生产提供机制基础。 公共卫生相关性：全球 50% 以上的人类疾病，包括癌症和传染病，仅由粮食短缺造成的营养不良就造成了。粮食生产的主要限制是全球范围内淡水资源的稀缺，目前超过 70% 的淡水用于农业。解决这一关键问题的一个方法是提高作物的水分利用效率，但实现这一解决方案的一个关键障碍是我们对植物应对水分胁迫的分子机制了解甚少。该项目通过研究脱落酸（ABA）的信号机制来解决这一关键问题，脱落酸是植物应对水分胁迫的中央调节剂。