Structural features of the nuclear receptor signaling code

核受体信号编码的结构特征

• 批准号: 8727622
• 负责人: Kendall W Nettles
• 金额: $ 90万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727622
• 关键词: Address; Anti-Inflammatory Agents; Antineoplastic Agents; Architecture; Bile Acids; Binding Sites; Biochemistry; Biological; Biological Assay; Biology; Boxing; Cell physiology; Cells; Cholesterol; Code; Communication; Complex; Crystallization; Crystallography; DNA; Deuterium; Diabetes Mellitus; Endocrine; Endocrinology; Enzymes; Family; Fatty Acids; Gene Expression; Goals; Human; Hydrogen; Individual; Inflammation; Length; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Nature; Nuclear Receptors; Outcome; Peptides; Physiology; Protein Structure Initiative; Proteins; Publishing; Reading; Receptor Signaling; Recruitment Activity; Retinoids; Robotics; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Molecule; Steroid Receptors; Stream; Structure; Testing; Thyroid Hormones; United States National Institutes of Health; Validation; Vitamin D; Work; base; birth control; combinatorial; design; drug discovery; drug structure; expression cloning; flexibility; protein complex; protein expression; scaffold; screening; small molecule; structural genomics; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define structural rules that govern how the nuclear receptor (NR) superfamily of 48 human transcription factors impact mammalian physiology. There is currently very little known about the structural features that allow small molecule NR ligands to induce specific signaling outcomes, and to date there are only structures of NR sub-regions and domains. We hypothesize that this nuclear receptor (NR) signaling code is defined by interdomain communication with the NR, and its modulation by specific interacting proteins, which are also the effectors of NR signaling, such as transcriptional co-regulator enzymes. It is critical to study complexes both because 1) they are the physiologically relevant structure for a signaling scaffold protein; and 2) co-regulator proteis can stabilize unstructured regions or interdomain interactions and facilitate obtaining structural information. To date, there is only one multi-domain NR complex characterized by x-ray crystallography. A significant barrier to obtaining structures of multi-domain NR constructs has been the conformational flexibility in the different domains, which has been solved for individual domains by addition of interacting molecules, including ligands, DNA, and small peptides. Importantly, only a small subset of interacting molecules are efficient in promoting crystallization, requiring the ability to assay many permutations. A PSI biology consortium is uniquely designed to test many permutations of cloning, expression, and crystallization. Our unique contribution is a high throughput NR drug discovery and structure based design platform, which affords us the expertise to both define optimal interaction molecules and complexes, but also the diversity of approaches for functional validation of resulting structures. We break up the work into two target streams: Target Stream 1 represents the full length NRs, which can be rapidly and directly submitted to the collaborative PSI center. We will work closely with the PSI center to implement a collaborative high throughput platform for salvage and optimization of protein expression. We will use our unique robotic Cell Based Screening Core to implement high throughput biochemistry assays to characterize protein quality, and to define optimal combinations of interacting molecules to promote crystallization, including ligands, DNA, and small NR box peptides. Target Stream 2 focuses on NR complexes. Starting from the list of 350 published NR coregulators, we will characterize those that directly interact with unstructured regions or multiple NR domains, and optimize complex architecture for crystallization for submission to the PSI center. The resulting structures will serve as the basis for functional studies on interdomain communication, allostery, and NR-complex signaling pathways, thus completing the loop in using structure to delineate the nuclear receptor signaling code. Importantly, the proteins and complexes produced by the PSI center will also be used to define structural features of allosteric signaling using our automated high throughput platform for assaying conformational dynamics with hydrogen/deuterium exchange mass spectrometry (HDX).

描述（由申请人提供）：本提案的总体目标是定义控制48种人类转录因子的核受体（NR）超家族如何影响哺乳动物生理学的结构规则。目前，关于允许小分子NR配体诱导特异性信号传导结果的结构特征知之甚少，并且迄今为止仅存在NR亚区和结构域的结构。我们假设，这种核受体（NR）信号传导代码是由与NR的域间通信定义的，并且其由特定的相互作用蛋白质进行调节，这些蛋白质也是NR信号传导的效应子，例如转录辅助调节酶。研究复合物是至关重要的，因为1）它们是信号支架蛋白的生理相关结构; 2）共调节蛋白可以稳定非结构化区域或结构域间相互作用，并有助于获得结构信息。迄今为止，只有一个多域NR复合物的特征在于通过X射线晶体学。获得多结构域NR构建体的结构的一个重要障碍是不同结构域中的构象灵活性，这已经通过添加相互作用的分子（包括配体、DNA和小肽）来解决各个结构域。重要的是，只有一小部分相互作用的分子在促进结晶方面是有效的，这需要测定许多排列的能力。一个PSI生物学联盟是独特的设计，以测试克隆，表达和结晶的许多排列。我们的独特贡献是高通量NR药物发现和基于结构的设计平台，这为我们提供了定义最佳相互作用分子和复合物的专业知识，以及对所得结构进行功能验证的方法的多样性。我们打破了 分为两个目标流：目标流1表示全长NR，其可以快速且直接地提交到协作PSI中心。我们将与PSI中心密切合作，实施一个协作的高通量平台，用于挽救和优化蛋白质表达。我们将使用我们独特的机器人Cell Based Screening Core来实施高通量生物化学分析，以表征蛋白质质量，并确定相互作用分子的最佳组合，以促进结晶，包括配体，DNA和小NR盒肽。目标流2关注NR复合物。从350个已发表的NR共调节子的列表开始，我们将表征那些直接与非结构化区域或多个NR结构域相互作用的共调节子，并优化复杂的结晶结构，以提交给PSI中心。由此产生的结构将作为域间通信，变构和NR-复合物信号通路的功能研究的基础，从而完成使用结构来描绘核受体信号编码的循环。重要的是，PSI中心产生的蛋白质和复合物也将用于定义变构信号的结构特征，使用我们的自动化高通量平台，用氢/氘交换质谱法（HDX）测定构象动力学。