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.
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