Structural dynamics of progesterone receptor-coactivator complexes

黄体酮受体-辅激活剂复合物的结构动力学

• 批准号: 10626857
• 负责人: Dean P Edwards
• 金额: $ 59.67万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-24 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626857
• 关键词: 3-Dimensional; Affect; Agonist; Attenuated; Binding; Biological Assay; Biology; Cells; Classification; Clinical; Complex; Coupled; Crosslinker; Cryoelectron Microscopy; DNA; DNA receptor; Data; Deuterium; Development; Drug Interactions; EP300 gene; Electrons; Environment; Exhibits; Gene Activation; Genes; Genetic Transcription; Goals; Heterogeneity; Hydrogen; Image; Kinetics; Knowledge; Length; Ligands; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Measures; Mediating; Medicine; Modeling; Molecular Conformation; Mutagenesis; Mutate; Mutation; N-terminal; NCOA3 gene; Negative Staining; Normal tissue morphology; Nuclear; Outcome; Pathologic; Pharmaceutical Preparations; Phase; Physiological; Process; Progesterone Receptors; Progestins; Protein Hormone Receptor; Protein Isoforms; Proteins; RXR; Reporting; Resolution; Response Elements; Role; Structural Models; Structure; Surface; Techniques; Therapeutic; Tissues; Transactivation; Transcription Coactivator; X-Ray Crystallography; base; crosslink; data exchange; detector; experimental study; flexibility; image processing; insight; nanometer resolution; next generation; novel; progesterone receptor A; progesterone receptor B; protein complex; receptor; reconstruction; response; screening; steroid hormone receptor; three dimensional structure; tool; transcription factor

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.

摘要：类固醇激素受体 (SR) 是配体依赖性核转录因子，表现出 介导细胞/组织和靶基因特异性反应的显着功能多样性，很大程度上是由 SR 蛋白的构象动力学，使其能够与转录辅酶的独特子集结合 调节蛋白 (CoR) 和 DNA 反应元件。黄体酮受体（PR）是主要靶点 临床上广泛使用的孕激素。 PR 表达为两种蛋白质亚型，一种是 N 端截短的 PR-A 和全长 PR-B 各自具有不同的生理作用，具体取决于细胞/组织类型。一般来说 PR-A 是比 PR-B 弱的转录激活剂，可以减弱 PR-B 的活性。两种异构体 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 据报道，病理状况变化很大。活性差异的机制基础 同种型尚未明确定义，但通常认为是由于结构上未知的差异造成的 构象。因此，要充分了解 PR 的生物学特性，需要确定 PR 的高分辨率结构。 全长 PR 异构体和相关 CoR 作为目标 DNA 上的复合物，并了解蛋白质如何 复杂和结构构象内的相互作用影响 PR 的活性。构象灵活性 SR 和 CoR 的耦合及其大尺寸 (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，该提案将整合补充解决方案 - 确定高分辨率 3D 结构模型并揭示构象动力学的相技术 PR:CoR/DNA 复合物内。冷冻电镜的最新进展使得能够确定溶液相 亚纳米分辨率的大型构象异质大分子复合物的结构。我们 将冷冻电镜与交联质谱 (XL-MS) 相结合，进一步完善结构冷冻电镜模型 并确保高分辨率并通过氢-氘交换 (HDX) 来绘制构象动力学和 PR:CoR/DNA 复合物内的变构。该项目的总体目标是确定最高分辨率 全长 PR-A 和 PR-B 与 PR DNA 上的经典 CoR 和新型 CoR 复合物的 3D 结构 响应元素。目标 1 将利用 Cryo-EM 分析复合物中 PR-A 和 PR-B 的结构特征 经典的 CoRs SRC3 和 p300 以及新颖的 CoRs TBP 和 JDP2 在目标 DNA 上组装。 目标 2 将使用集成结构建模和 XL- 完善 PR:CoR/DNA 复合物的冷冻电镜结构 MS 通过差分 HDX 定义距离约束并探测 PR 复合物内的构象动力学。 目标 3 将进行功能诱变研究，以确定 PR:SRC3/p300 相互作用的影响 结构模型和 XL-MS 数据中显示的表面对 PR 活性有影响。该提案的影响 将填补我们对 PR:CoR/DNA 结构和构象动力学理解的重大空白 复杂的。这些研究可能为原子水平上药物相互作用的新研究提供机会。