Abstract
The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.
Highlights
The Aryl hydrocarbon Receptor (AhR) is a basic Helix-Loop-Helix PER-AhR Nuclear Translocator (ARNT)-SIM-containing transcription factor that responds to a variety of structurally diverse exogenous and endogenous chemicals with the modulation of gene expression and production of diverse biological and toxic effects in a wide range of species and tissues [1]
Homology modeling of the murine AhR:ARNT PAS domain dimers was performed adopting the following X-ray structures of mammalian PAS domain dimers: the human HIF2α:ARNT PAS-B heterodimer (PDB 3F1P) [11]; the murine CLOCK:BMAL1 heterodimer, inclusive of the bHLH, PAS-A and PAS-B domains (PDB 4F3L) [13]; and the murine AhR PAS-A homodimer (PDB 4M4X) [12] (Fig 1)
Different models of the PAS-A and PAS-B dimers were assembled from the protomer models, according to the different dimerization modes described by the template structures
Summary
The Aryl hydrocarbon Receptor (AhR) is a basic Helix-Loop-Helix PER-ARNT-SIM (bHLH-PAS)-containing transcription factor that responds to a variety of structurally diverse exogenous and endogenous chemicals with the modulation of gene expression and production of diverse biological and toxic effects in a wide range of species and tissues [1]. Recent studies have revealed endogenous regulatory roles for the AhR in normal physiology and development, including the pivotal role that this ligand-dependent transcription factor plays in the differentiation and/or affinity maturation of several key immune cells important in both innate and acquired immune response [1]. These findings have generated a renewed interest in understanding the molecular mechanisms of activation and their contribution to different complex physiological roles of the receptor [1,2,3]. Binding of the ligand:AhR:ARNT complex to its specific DNA recognition site, the Dioxin Responsive Element (DRE), stimulates transcription of adjacent genes and the production of the spectrum of biological and toxic effects of AhR ligands [1,3]
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