Structural features of the nuclear receptor signaling code

描述(申请人提供)：这项提案的总体目标是定义结构规则，该规则管理由48个人类转录因子组成的核受体(NR)超家族如何影响哺乳动物的生理。目前对小分子NR配体诱导特定信号转导结果的结构特征知之甚少，到目前为止只有NR亚区和结构域的结构。我们推测这种核受体(NR)信号编码是由核受体与核受体之间的跨域通信以及特定相互作用蛋白的调控所定义的，这些蛋白也是核受体信号的效应者，如转录共调节酶。研究复合体是至关重要的，因为1)它们是信号支架蛋白的生理相关结构；2)协同调节蛋白可以稳定非结构区域或结构域间的相互作用，并有助于获得结构信息。到目前为止，用X射线结晶学表征的多结构域天然橡胶络合物只有一个。获得多域NR结构的一个重要障碍是不同结构域的构象灵活性，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。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，这需要测试许多排列的能力。PSI生物联盟是专门为测试克隆、表达和结晶的许多排列而设计的。我们的独特贡献是一个高通量的NR药物发现和基于结构的设计平台，该平台为我们提供了定义最佳相互作用分子和络合物的专业知识，以及对所产生的结构进行功能验证的多样化方法。我们打碎了 分成两个目标流：目标流1代表全长NRS，可以快速直接提交到协同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，用于打捞和优化蛋白质表达。我们将使用我们独特的基于机器人细胞的筛选核心来实现高通量生化分析，以表征蛋白质质量，并确定相互作用的分子的最佳组合，以促进结晶，包括配体、DNA和小NR盒肽。目标流2专注于天然橡胶复合体。从已公布的350个NR协调控子的列表开始，我们将描述那些直接与非结构化区域或多个NR结构域相互作用的结构，并优化复杂的架构以结晶提交给PSI中心。所得到的结构将为跨结构域通讯、变构和NR-复合体信号通路的功能研究奠定基础，从而完成利用结构来描述核受体信号编码的循环。重要的是，PSI中心生产的蛋白质和复合体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/氢交换质谱仪(HDX)分析构象动力学。

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).