Structural dynamics of progesterone receptor-coactivator complexes

概述：类固醇激素受体（SR）是配体依赖性核转录因子， 介导细胞/组织和靶基因特异性应答的显著功能多样性，主要由以下因素驱动： SR蛋白的构象动力学，使其能够结合独特的转录辅基亚群， 调节蛋白（CoRs）和DNA反应元件。孕激素受体（PR）是 临床上广泛使用的孕激素。PR表达为两种蛋白质同种型，N-末端截短型和N-末端截短型。 PR-A和全长PR-B各自具有取决于细胞/组织类型的不同生理作用。一般来说 PR-A是比PR-B弱的转录激活因子，并且可以起到减弱PR-B的活性的作用。两种同种型 通常在大多数正常组织中以相等的比例共表达。然而，PR-A与PR-B的比率一直是 据报道，在病理条件下高度可变。活性差异的机制基础 同种型没有很好地定义，但通常认为是由于结构上的未知差异 构象因此，为了充分理解PR的生物学，需要确定PR的高分辨率结构。 全长PR异构体和相关的CoRs作为靶DNA上的复合物，以及对蛋白质如何 复合物和结构构象内的相互作用影响PR的活性。 SR和CoR的耦合和它们的大尺寸（100-300 MW）使它们不适合高分辨率 NMR或X射线晶体学分析。作为替代方案，本提案将整合补充解决方案- 相位技术，以确定高分辨率的3D结构模型，并揭示构象动力学 PR：CoR/DNA复合物Cryo-EM的最新进展使溶液相的测定成为可能 结构的大构象异质大分子复合物在亚纳米分辨率。我们 将联合收割机Cryo-EM与交联质谱（XL-MS）相结合，进一步完善Cryo-EM结构模型 并确保高分辨率和氢氘交换（HDX）映射构象动力学， PR：CoR/DNA复合物内的变构。本项目的总体目标是确定最高分辨率 PR-A和PR-B全长与PR DNA上的经典CoR和新型CoR复合的3D结构可能 响应元素。目的1利用冷冻电镜技术分析复合物中PR-A和PR-B的结构特征 与经典的CoRs SRC 3和p300以及与组装在靶DNA上的新型CoRs TBP和JDP 2。 目的2将使用集成的结构建模和XL-1优化PR：CoR/DNA复合物的Cryo-EM结构。 MS通过差分HDX定义PR复合物内的距离约束和探针构象动力学。 目的3将进行功能性诱变研究，以确定PR：SRC 3/p300相互作用的影响 结构模型和XL-MS数据显示的表面对PR活性有影响。这项提议的影响 将填补我们对PR：CoR/DNA结构和构象动力学理解的一个主要空白 复杂.这些研究可能为在原子水平上进行药物相互作用的新研究提供机会。

Summary: Steroid hormones receptors (SR) are ligand-dependent nuclear transcription factors that exhibit remarkable functional diversity in mediating cell/tissue and target gene specific responses, largely driven by conformational dynamics of the SR protein that enables it's binding of unique subsets of transcriptional co- regulatory proteins (CoRs) and DNA response elements. The progesterone receptor (PR) is the main target of progestogens that are widely used clinically. PR is expressed as two protein isoforms, an N-terminal truncated PR-A and full-length PR-B and each have distinct physiological roles dependent on the cell/tissue type. In general PR-A is a weaker transcriptional activator than PR-B, and can act to attenuate the activity of PR-B. Both isoforms are typically co-expressed in equal proportions in most normal tissues. However, PR-A to PR-B ratios have been reported to be highly variable in pathological conditions. Mechanistic basis for differences in activity of the isoforms is not well defined but is generally believed to be due to unknown differences in structural conformations. Thus, to fully understand PRs' biology requires determination of a high-resolution structure of the full-length PR isoforms and associated CoRs as a complex on target DNA and an understanding of how protein interactions within the complex and structural conformations affect activity of PR. The conformational flexibility of SRs and CoRs, coupled and their large sizes (100–300 MW), make them unsuitable to either high resolution NMR or X-ray crystallography analysis. As an alternative, this proposal will integrate complementary solution- phase techniques to determine high-resolution 3D structural models and uncover the conformational dynamics within the PR:CoR/DNA complex. Recent advances in Cryo-EM enable the determination of solution-phase structures of large conformationally heterogenous macromolecular complexes at subnanometer resolution. We will combine Cryo-EM with crosslinking mass spectrometry (XL-MS) to further refine structural Cryo-EM models and assure high resolution and with hydrogen-deuterium exchange (HDX) to map conformational dynamics and allostery within the PR:CoR/DNA complex. The overall goal of this project is to determine the highest resolution 3D structure possible of full-length PR-A and PR-B in complex with classical CoRs and novel CoRs on PR DNA response elements. Aim 1 will utilize Cryo-EM to analyze the structural features of PR-A and PR-B in complex with the classical CoRs SRC3 and p300 and with the novel CoRs TBP and JDP2 assembled on target DNA. Aim 2 will refine the Cryo-EM structure of PR:CoR/DNA complex using integrated structural modeling and XL- MS to define distance constraints and probe conformational dynamics within the PR complex by differential HDX. Aim 3 will perform functional mutagenesis studies to determine the influence of PR:SRC3/p300 interaction surfaces revealed in structural models and from XL-MS data have on PR activity. The impact of this proposal will be to fill a major gap in our understanding of the structure and conformational dynamics of the PR:CoR/DNA complex. These studies could open opportunities for novel studies of drug interactions at the atomic level.