Analysis of Ah Receptor Ligand Binding Specificity

描述（申请人提供）：Ah受体（AhR）是一种配体依赖性转录因子，介导多种化学品的毒性和生物学效应，据信这些效应是易感细胞基因表达的受体依赖性改变所致。虽然AhR的最佳特征和最高亲和力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具有混杂的配体结合结构域（LED），并提出了关于可以结合和/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配体的反应中起关键的关键作用，但关于AhR LED内这些配体的特异性结合相互作用或这些相互作用如何导致AhR和AhR信号转导的配体依赖性激活的信息很少。我们假设，AhR配体和AhR LBD内包含的关键氨基酸之间的特异性结合相互作用是负责差异配体亲和力和特异性以及配体诱导的AhR LBD结构的变化，导致AhR激活。由于缺乏AhR LBD的三维模型，以前无法验证这一假设。然而，我们最近开发的PAS结构域的AhR LBD的同源性模型提供了一个途径，通过该途径进行这些迄今未定义的事件的机械定向结构分析。因此，我们建议开发，优化和验证的AhR LBD的结构的同源性模型的基础上现有的晶体结构的PAS结构域从相关蛋白质。将使用定点诱变和突变体AhR的AhR功能活性的表征来确认模型的验证。定量构效关系和分子对接的方法将被用来模拟特定的结合配体内的建模AhR LBD，并将利用结合数据获得的功能分析库的新的黄酮类化合物和靛玉红。定点诱变和AhR功能研究也将与建模方法结合使用，以鉴定和表征负责与hsp 90相互作用的AhR和AhR LBD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的分析，但他们将提供新的见解观察到的物种和配体特异性结合和激活的AhR的差异。

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.