Analysis of Ah Receptor Ligand Binding Specificity

Ah 受体配体结合特异性分析

• 批准号: 8975770
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 30.77万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8975770
• 关键词: 3-Dimensional; Affinity; Agonist; Amino Acids; Antineoplastic Agents; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Binding; Binding Sites; Biological; Characteristics; Chemicals; Client; Collection; Complex; Coupled; DNA Binding; Development; Developmental Process; Dioxins; Docking; Exhibits; Fishes; Gene Expression; Goals; Homology Modeling; Human; Lead; Ligand Binding; Ligand Binding Domain; Ligands; Measurement; Mediating; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Mus; Mutation; Pathway interactions; Physiological; Physiological Processes; Process; Proteins; Proteolysis; Protocols documentation; Receptor Activation; Receptor Signaling; Reporter Genes; Reporting; Roentgen Rays; Role; Signal Pathway; Site-Directed Mutagenesis; Specificity; Structure; Testing; Therapeutic; Toxic effect; Toxin; Update; Work; base; conformational conversion; induced pluripotent stem cell; inhibitor/antagonist; insight; molecular dynamics; pregnane X receptor; protein structure; receptor; receptor binding; transcription factor

DESCRIPTION (provided by applicant): The Ah receptor (AhR) is a ligand-dependent transcription factor known to regulate the toxic and biological effects of a variety of exogenous chemicals, such as the toxic halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs), and these effects appear to result from AhR-dependent alterations in gene expression. The AhR is also involved in several endogenous developmental and physiological processes, although the responsible endogenous ligand(s) is unknown. While HAHs and PAHs are the prototypical and highest affinity ligands, the AhR can bind and be activated by a diverse range of structurally dissimilar compounds, even though species- and ligand-specific differences in AhR ligand binding specificity and functionality exist. Site-directed mutagenesis and functional analysis studies based on our 3-dimensional (3D) homology model of the AhR ligand binding domain (LBD) performed so far have allowed initial understanding of aspects of the process by which high affinity HAH ligands like 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) can bind to and activate the AhR. However, these same studies suggest that significant differences exist in the amino acid residues to which structurally unrelated AhR ligands specifically interact. We hypothesize that differences in the binding sites and interactions of structurally diverse AhR ligands within the AhR LBD are primarily responsible for the observed ligand promiscuity of the AhR and that these differences could contribute to ligand-specific alterations in AhR conformational states that lead to differences in AhR functionality. To test this hypothesis, we propose to develop a new homology model of the AhR LBD based on recently released X-ray structures of the HIF-2a template complexed with ligands that also bind to the AhR and use this updated model for docking analysis of AhR ligands. Structurally driven site-directed mutagenesis and AhR functional analysis of the interactions of structurally diverse AhR agonists/antagonists with specific amino acids within the AhR LBD, coupled with similar analyses of chimeric mouse AhRs containing the LBD domain of AhRs which do not bind TCDD, will facilitate further identification of residues and structural characteristics of the LBD required for ligand binding. The molecular mechanisms by which binding of structurally diverse ligands within the LBD stimulates transformation of the AhR into its DNA binding form (loss of hsp90 and binding of Arnt) will be examined through analysis of AhR:hsp90 and AhR:Arnt interactions and ligand selective effects on coactivator binding and AhR-dependent gene expression determined. The residues/regions of both proteins involved in complex formation and functional activity will be defined and modeled and ligand-specific alterations in these mechanisms examined. The studies proposed here will provide detailed analysis of the molecular mechanisms by which structurally diverse ligands bind to and activate the AhR. In addition, they will yield insights into the molecular mechanisms of ligand-dependent AhR transformation, the influence of ligand structure on these processes and the diversity of AhR responsiveness.

描述（由申请人提供）：Ah受体（AhR）是一种配体依赖性转录因子，已知可调节多种外源化学物质的毒性和生物效应，例如有毒的卤代芳香烃（HAH）和多环芳香烃（PAH），并且这些效应似乎是由AhR依赖性基因表达改变引起的。 AhR 还参与一些内源性发育和生理过程，尽管负责的内源性配体尚不清楚。虽然 HAH 和 PAH 是原型和最高亲和力的配体，但 AhR 可以结合多种结构不同的化合物并被其激活，即使 AhR 配体结合特异性和功能性存在物种和配体特异性差异。迄今为止，基于我们的 AhR 配体结合域 (LBD) 的 3 维 (3D) 同源模型进行的定点诱变和功能分析研究，使我们能够初步了解高亲和力 HAH 配体（如 2,3,7,8-四氯二苯并-对二恶英 (TCDD)）可以结合并激活 AhR 的过程的各个方面。然而，这些相同的研究表明，结构上不相关的 AhR 配体特异性相互作用的氨基酸残基存在显着差异。我们假设，AhR LBD 内结构多样的 AhR 配体的结合位点和相互作用的差异是观察到的 AhR 配体混杂性的主要原因，并且这些差异可能导致 AhR 构象状态的配体特异性改变，从而导致 AhR 功能的差异。为了检验这一假设，我们建议基于最近发布的与也与 AhR 结合的配体复合的 HIF-2a 模板的 X 射线结构，开发一种新的 AhR LBD 同源模型，并使用此更新的模型对 AhR 配体进行对接分析。对结构多样的AhR激动剂/拮抗剂与AhR LBD内特定氨基酸的相互作用进行结构驱动的定点诱变和AhR功能分析，再加上对含有不结合TCDD的AhR的LBD结构域的嵌合小鼠AhR的类似分析，将有助于进一步鉴定配体结合所需的LBD的残基和结构特征。通过分析 AhR:hsp90 和 AhR:Arnt 相互作用以及配体对共激活剂结合和 AhR 依赖性基因表达的选择性影响，将检查 LBD 内结构不同的配体结合刺激 AhR 转化为其 DNA 结合形式（hsp90 丢失和 Arnt 结合）的分子机制。涉及复合物形成和功能活性的两种蛋白质的残基/区域将被定义和建模，并检查这些机制中的配体特异性改变。这里提出的研究将对结构多样的配体结合并激活 AhR 的分子机制进行详细分析。此外，他们还将深入了解配体依赖性 AhR 转化的分子机制、配体结构对这些过程的影响以及 AhR 反应的多样性。