Analysis of Ah Receptor Ligand Binding Specificity

描述(申请人提供)：ah受体(AhR)是一种配体依赖的转录因子，已知调节各种外源化学物质的毒性和生物效应，如有毒的卤代芳香烃(HAHS)和多环芳烃(PAHs)，这些效应似乎是依赖AhR的基因表达变化的结果。AhR也参与了几个内源性的发育和生理过程，尽管内源配体(S)的作用尚不清楚。虽然HAHS和PAHs是典型的和最高亲和力的配体，但AhR可以与一系列结构不同的化合物结合并被激活，尽管不同物种和配体在AhR配体结合特异性和功能上存在差异。到目前为止，基于我们的AhR配体结合域(LBD)的三维(3D)同源模型进行的定点突变和功能分析研究，使得人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)与AhR结合并激活AhR的过程。然而，这些相同的研究表明，结构上不相关的AhR配体具体与之相互作用的氨基酸残基存在显著差异。我们假设，AhR LBD内结构不同的AhR配体的结合部位和相互作用的差异是观察到的AhR配体混杂的主要原因，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。为了验证这一假设，我们建议基于最近发布的HIF-2a模板与也与AhR结合的配体的X射线结构，开发一个新的AhR LBD同源模型，并使用这个更新的模型对AhR配体进行对接分析。结构驱动的定点突变和AhR功能分析不同的AhR激动剂/拮抗剂与AhR LBD内特定氨基酸的相互作用，加上对含有AHRS的LBD结构域的嵌合小鼠AHRs的类似分析(不与TCDD结合)，将有助于进一步鉴定配体结合所需的LBD残基和结构特征。通过分析AhR：HSP90和AhR：Arnt的相互作用和配体选择性对辅活子结合和AhR依赖的基因表达的影响，将研究LBD内结构不同的配体结合刺激AhR转变为DNA结合形式(HSP90丢失和Arnt结合)的分子机制。将对参与复合体形成和功能活性的两种蛋白质的残基/区域进行定义和建模，并研究这些机制中的配体特异性变化。这些研究将详细分析结构不同的配体与AhR结合并激活AhR的分子机制。此外，他们还将深入了解配体依赖的AhR转化的分子机制，配体结构对这些过程的影响，以及AhR响应的多样性。

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.