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.
Antibody-antigen recognition plays a key role in the immune response against pathogens. Here, we have investigated various aspects of this problem by analyzing a large and diverse set of antibodies and their respective complexes with protein antigens through atomistic simulations. Common features of antibody response to the presence of antigens are elucidated by the analysis of the proteins' internal dynamics and coordination in different ligand states, combined with the analysis of the interaction networks implicated in the stabilization of functional structures. The use of a common structural reference reveals preferential changes in the dynamic coordination and intramolecular interaction networks induced by antigen binding and shared by all antibodies. Such changes propagate from the binding region through the whole immunoglobulin domains. Overall, complexed antibodies show more diffuse networks of nonbonded interactions and a general higher internal dynamic coordination, which preferentially involve the immunoglobulin (Ig) domains of the heavy chain. The combined results provide atomistic insights into the correlations between the modulation of conformational dynamics, structural stability, and allosteric signal transduction. In particular, the results suggest that specific networks of residues, shared among all the analyzed proteins, define the molecular pathways by which antibody structures respond to antigen binding. Our studies may have implications in practical use, such as the rational design of antibodies with specifically modulated antigen-binding affinities.
Molecular modeling has given important contributions to elucidation of the main stages in the AhR signal transduction pathway. Despite the lack of experimentally determined structures of the AhR functional domains, information derived from homologous systems has been exploited for modeling their structure and interactions. Homology models of the AhR PASB domain have provided information on the binding cavity and contributed to elucidate species-specific differences in ligand binding. Molecular Docking simulations of the ligand binding process have given insights into differences in binding of diverse agonists, antagonists, and selective AhR modulators, and their application to virtual screening of large databases of compounds have allowed identification of novel AhR ligands. Recently available structural information on protein-protein and protein-DNA complexes of other bHLH-PAS systems has opened the way for modeling the AhR:ARNT dimer structure and investigating the mechanisms of AhR transformation and DNA binding. Future research directions should include simulation of the protein dynamics to obtain a more reliable description of intermolecular interactions involved in signal transmission.
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