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: 8500400
• 负责人: Karsten Melcher
• 金额: $ 34.84万
• 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/8500400
• 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.

描述（由申请人提供）：大多数信号通路涉及不稳定的动态蛋白质复合物，这些蛋白质复合物迅速分离，因此，通过高分辨率结构研究很难分析它们。在此提案中，我们将使用蛋白质工程来确定关键植物胁迫激素脱离的脱节酸（ABA）的动态信号传导复合物的晶体结构。 ABA是一种古老的信号分子，在植物，真菌和后生动物中发现，从海绵到人类。在植物中，ABA是一种必不可少的激素，也是保护植物免受干旱，冷和盐分等非生物胁迫的中心调节剂。这些应力是作物生产的主要限制因素，因此由于食物短缺而导致营养不良的主要因素。这与人类健康有关，因为营养不良促成了全球超过50％的人类疾病，包括癌症和传染病。了解ABA信号传导的详细机制对于为植物中ABA途径的基因工程提供机械基础至关重要。在ABA信号传导的中心是与AMPK相关的蛋白激酶家族，通过磷酸化转录因子，离子通道和第二届年龄段产生酶来中继ABA信号。这些激酶在2C型蛋白磷酸酶（PP2C）和细胞内ABA受体的控制之下。在此提案中，提出了含有受体，ABA，PP2CS和SNRK2的第四纪信号传导复合物的存在。我们将使用蛋白质工程来稳定这些复合物，并使其适合X射线晶体学。这些复合物的结构将为这些复合物的功能提供重要的见解，并将在复合物的背景下确定所有蛋白质 - 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中以及转基因植物中体内的功能。该项目的结果将为ABA信号传导中对受体，ABA，PP2C和SNRK2相互作用的结构理解提供一个综合框架，因此将为调节植物中的ABA途径提高其水利用效率和食品生产提供机械基础。