Structural dynamics of progesterone receptor-coactivator complexes

摘要：类固醇激素受体(SR)是配体依赖的核转录因子，表现为 介导细胞/组织和靶基因特异性反应的显著功能多样性，主要由 SR蛋白的构象动力学，使其能够与转录辅助蛋白的独特亚集结合 调节蛋白(CORs)和DNA反应元件。孕激素受体(PR)是肿瘤的主要靶点 临床上广泛使用的促孕素。PR表达为两种蛋白质亚型，N端截短 PR-A和全长PR-B各自具有不同的生理作用，取决于细胞/组织类型。总体而言 PR-A是一种弱于PR-B的转录激活剂，可以减弱PR-B的活性。两种异构体 在大多数正常组织中通常以相同比例共表达。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。人类活动差异的机制基础 异构体的定义不是很清楚，但通常认为是由于结构上的未知差异 构象。因此，要充分了解PR的生物学特性，需要确定其高分辨率结构 全长PR异构体和相关CORs作为靶DNA上的复合体以及对蛋白质如何 复杂构象和结构构象之间的相互作用影响PR的活性。构象柔性 SRS和CORS的组合，以及它们的大尺寸(100-300 mW)，使得它们不适合任何一种高分辨率 核磁共振或X射线结晶学分析。作为替代方案，该提案将整合互补的解决方案- 确定高分辨率三维结构模型和揭示构象动力学的相技术 在PR中：COR/DNA复合体。低温电子显微镜的最新进展使溶液相的测定成为可能 亚纳米分辨率下构象多相大分子络合物的结构。我们 将把Cryo-EM与交联质谱(XL-MS)相结合，以进一步完善结构Cryo-EM模型 并确保高分辨率并使用氢-氚交换(HDX)来绘制构象动力学图和 PR中的变构：COR/DNA复合体。该项目的总体目标是确定最高分辨率 PR DNA上经典CORs和新型CORs与全长PR-A和PR-B形成的复合体的三维结构 响应要素。目标1将利用冷冻-EM分析复合体中PR-A和PR-B的结构特征 与经典的CORS SRC3和P300以及新型CORS TBP和JDP2组装在靶DNA上。 目标2将使用集成结构建模和XL-EM来提炼PR：COR/DNA复合体的冷冻-EM结构 MS来定义距离限制，并通过差分HDX探测PR复合体内的构象动力学。 AIM 3将进行功能突变研究，以确定PR：SRC3/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的结构和构象动力学的一个主要空白：COR/DNA 很复杂。这些研究可能为在原子水平上研究药物相互作用提供新的机会。

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.