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，介导多种化学物质的毒性和生物学效应，据信这些效应是由易感细胞中受体依赖性基因表达改变引起的。虽然AhR的最佳表征和亲和力最高的配体包括各种有毒的卤代芳烃，但最近的研究表明，AhR可以被各种结构不同的化学物质激活。这一观察结果强烈表明，AhR具有混杂配体结合域（LED），并提出了有关能够结合和/或激活AhR的化学物质的实际光谱的问题。虽然很明显，AhR在介导细胞对AhR配体的反应中起着关键的关键作用，但关于AhR LED中这些配体的具体结合相互作用或这些相互作用如何导致AhR的配体依赖性激活和AhR信号转导的信息很少。我们假设AhR配体与AhR LBD中包含的关键氨基酸之间的特异性结合相互作用是导致不同配体亲和力和特异性以及配体诱导的AhR LBD结构变化导致AhR活化的原因。由于缺乏AhR LBD的三维模型，以前不可能对这一假设进行检验。然而，我们最近开发的AhR LBD PAS结构域的同源模型提供了一种途径，通过该途径可以对这些迄今未定义的事件进行机械定向结构分析。因此，我们建议基于相关蛋白PAS结构域的现有晶体结构，开发、优化和验证AhR LBD结构的同源性模型。该模型的验证将通过位点定向诱变和突变AhR功能活性的表征来证实。QSAR和分子对接方法将用于模拟AhR lbd中配体的特异性结合，并将利用通过对新型类黄酮和靛红素库的功能分析获得的结合数据。位点定向诱变和AhR功能研究也将与建模方法结合使用，以识别和表征AhR和AhR LBD中负责与hsp90相互作用的区域，以及配体结合对这些相互作用的影响。总的来说，这里提出的研究不仅可以提供配体结合和激活AhR的机制的第一个详细分析，而且它们将为观察到的物种和配体特异性结合和激活AhR的差异提供新的见解。

DESCRIPTION (PROVIDED BY APPLICANT): The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals and it is believed that these effects result from receptor-dependent alterations in gene expression in susceptible cells. While the best characterized and highest affinity ligands for the AhR include a variety of toxic halogenated aromatic hydrocarbons, recent studies have revealed that the AhR can be activated by a wide variety of structurally dissimilar chemicals. This observation strongly suggest that the AhR has a promiscuous ligand binding domain (LED) and raises questions regarding the actual spectrum of chemicals which can bind to and/or activate the AhR. Although it's clear that that the AhR plays the key pivotal role in mediating the response of a cell to AhR ligands, there is little information with respect to the specific binding interactions of these ligands within the AhR LED or how these interactions lead to ligand-dependent activation of the AhR and AhR signal transduction. We hypothesize that specific binding interactions between AhR ligands and key amino acids contained within the AhR LBD are responsible for differential ligand affinity and specificity as well as ligand-induced changes in the AhR LBD structure that leads to AhR activation. Testing of this hypothesis has not been previously possible due to the lack of a 3- dimensional model of the AhR LBD. However, our recent development of a homology model of the PAS domain of the AhR LBD provides an avenue by which to carry out a mechanistically-directed structural analysis of these hitherto undefined events. Accordingly, we propose to develop, optimize and validate of homology model of the structure of the AhR LBD based on existing crystal structures for PAS domains from related proteins. Validation of the model will be confirmed using site-directed mutagenesis and characterization of AhR functional activities of mutant AhRs. QSAR and molecular docking approaches will be used to model the specific binding of ligands within the modeled AhR LBDs and will utilize binding data obtained by functional analysis of a library of novel flavonoids and indirubins. Site-directed mutagenesis and AhR functional studies will also be used in combination with modeling approaches to identify and characterize the regions of the AhR and AhR LBD responsible for interactions with hsp90 and the effect of ligand binding on these interactions. Overall, the studies proposed here will not only allow provide the first detailed analysis of the mechanisms by which ligands bind to and activate the AhR, but they will provide new insights into observed differences in species- and ligand-specific binding and activation of the AhR.