Analysis of Ah Receptor Ligand Binding Specificity

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性化学物质（如有毒卤代芳烃（HAH）和多环芳烃（PAH））的毒性和生物学效应，这些效应似乎是基因表达中AhR依赖性改变的结果。AhR还参与多种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存在物种特异性和配体特异性差异。定点突变和功能分析研究的基础上，我们的三维（3D）同源模型的AhR配体结合域（LBD）进行到目前为止，允许初步了解的过程中，高亲和力的HAH配体，如2，3，7，8-四氯二苯并-p-二恶英（TCDD）可以结合和激活AhR的方面。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，在AhR LBD内的结构不同的AhR配体的结合位点和相互作用的差异是主要负责观察到的配体混杂的AhR，这些差异可能有助于配体特异性改变AhR构象状态，导致AhR功能的差异。为了验证这一假设，我们建议开发一种新的同源性模型的AhR LBD的基础上最近发布的X射线结构的HIF-2a模板复合配体，也结合到AhR和使用这种更新的模型的AhR配体的对接分析。结构驱动的定点诱变和AhR功能分析的相互作用的结构不同的AhR激动剂/拮抗剂与特定的氨基酸内的AhR LBD，再加上类似的分析嵌合小鼠AhR含有LBD结构域的AhR不结合TCDD，将促进进一步鉴定的残基和结构特征的LBD所需的配体结合。将通过分析AhR：hsp 90和AhR：Arnt相互作用和配体选择性对辅激活因子结合和AhR依赖性基因表达的影响来研究LBD内结构多样的配体结合刺激AhR转化为其DNA结合形式（hsp 90损失和Arnt结合）的分子机制。这两种蛋白质的残基/区域参与复合物的形成和功能活性将被定义和建模，并检查这些机制中的配体特异性改变。这里提出的研究将提供详细的分析的分子机制，结构不同的配体结合和激活的AhR。此外，他们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。

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.