Structural and functional analysis of a dynamic ABA signaling complex

描述(申请人提供)：大多数信号通路涉及不稳定的、动态的蛋白质复合体，这些复合体迅速解离，因此出了名的难以通过高分辨率结构研究进行分析。在这个提案中，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(ABA)的动态信号复合体的晶体结构。ABA是一种古老的信号分子，存在于从海绵到人类的植物、真菌和后生动物中。在植物中，ABA是一种必需的激素，是保护植物免受干旱、寒冷和盐分等非生物胁迫的中枢调节因子。这些压力是作物生产的主要限制因素，因此是粮食短缺造成营养不良的主要因素。这与人类健康有关，因为营养不良占全球人类疾病的50%以上，包括癌症和传染病。了解ABA信号转导的详细机制对于为植物ABA信号转导途径的基因工程提供机制基础是至关重要的。ABA信号的中心是一系列AMPK相关的蛋白激酶，它们通过磷酸化转录因子、离子通道和第二信使产生酶来传递ABA信号。这些激酶受2C型蛋白磷酸酶(PP2Cs)和细胞内ABA受体的控制。在这一建议中，证据表明存在含有ABA、PP2Cs和SnRK2s受体的四元信号复合体。我们将使用蛋白质工程来稳定这些复合体，使它们符合X射线结晶学的要求。这些复合体的结构将为了解这些复合体的功能提供重要的见解，并将确定所有蛋白质-蛋白质和蛋白质-ABA相互作用的关键残基。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分析中的功能，以及在转基因植物中的体内功能。该项目的成果将为了解受体、ABA、PP2C和SnRK2在ABA信号中的相互作用提供一个全面的结构框架，从而为调节植物的ABA途径以提高其水分利用效率和食物生产提供机制基础。

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.