Structural features of the nuclear receptor signaling code

描述（由申请人提供）：本提案的总体目标是定义控制48个人类转录因子的核受体（NR）超家族如何影响哺乳动物生理的结构规则。目前对小分子NR配体诱导特定信号结果的结构特征知之甚少，迄今为止只有NR子区域和结构域的结构。我们假设这种核受体（NR）信号编码是由与NR的域间通信定义的，并由特定的相互作用蛋白调节，这些蛋白也是NR信号的效应器，如转录共调节酶。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域间相互作用，便于获取结构信息。迄今为止，只有一种多域NR配合物被x射线晶体学表征。获得多结构域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）来解决单个结构域的问题。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，这需要分析许多排列的能力。PSI生物学财团是唯一设计来测试克隆，表达和结晶的许多排列。我们独特的贡献是高通量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，同时也为结果结构的功能验证提供了多种方法。我们打破了

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).