Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

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.
Author Summary Computational modeling combined with experimental validation may give insight into structural and functional properties of protein systems. The basic Helix-Loop-Helix PER-ARNT-SIM (bHLH-PAS) proteins show conserved functional domains despite the broad range of functions exerted by the different systems. Within this protein family, the Aryl hydrocarbon Receptor (AhR) is known to mediate the toxic effects of a number of environmental contaminants, including dioxins and dioxin-like chemicals, and it also exerts other biochemical and physiological effects. Despite the absence of experimentally determined structures, theoretical models of the AhR PAS domains developed on the basis of homologous systems have allowed understanding of some aspects of the molecular mechanisms underlying its function. In this work we present alternative structural models of the transcriptionally active complex of AhR with the AhR Nuclear Translocator (ARNT) protein. Computational characterization of the modeled protein-protein interaction interfaces guided the design of mutagenesis experiments, and evaluation of the DNA binding ability of the resulting AhR:ARNT dimer mutants allowed validation of the models and selection of the most reliable one. These findings open new research directions for understanding the molecular mechanisms underlying the functional activity of the AhR.