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.
Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milan, Italy.
Dario
D
Corrada
Department of Environmental Toxicology, University of California, Davis, Davis, California, United States of America.
Anatoly A
AA
Soshilov
Department of Environmental Toxicology, University of California, Davis, Davis, California, United States of America.
Michael S
MS
Denison
Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milan, Italy.
Laura
0000-0003-3028-0368
L
Bonati
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D000072417:Protein Domains; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018336:Receptors, Aryl Hydrocarbon; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator