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Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses., Author summary A low oxygen condition, called hypoxia, often occurs in tumor masses and generally correlates with worse prognosis. Cells in a tumor react to low oxygen levels with a metabolism modification induced by the activation of hypoxia inducible factors (HIFs) through dimerization with a partner protein and binding to a DNA target. Disrupting this protein-protein interaction could be a potential therapeutic strategy, but directly interfering with dimer formation can be troublesome because of the difficulty to design drugs that bind to protein interfaces. However, ligands that bind internal protein cavities can indirectly perturb the interfaces reducing dimers stability. Albeit protein crystallography had offered a detailed static picture of a HIF dimer bound to candidate inhibitors, it is not able to describe either the perturbation caused by binding or the molecular mechanism of dimer destabilization. Here we exploit molecular dynamics to identify the crucial interfaces in the HIF dimer stabilization and, by comparing the results obtained in the bound and unbound forms, we reveal the mechanism of ligand inhibition at atomic detail. All these findings will guide toward the design of improved dimerization inhibitors, to contrast cell survival in tumor masses.

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.

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.

Unraveling the molecular basis of immune recognition still represents a challenging task for current biological sciences, both in terms of theoretical knowledge and practical implications. Here, we describe the physical-chemistry methods and computational protocols for the prediction of antibody-binding epitopes and MHC-II loaded epitopes, starting from the atomic coordinates of antigenic proteins (PDB file). These concepts are the base of the Web tool BEPPE (Binding Epitope Prediction from Protein Energetics), a free service that returns a list of putative epitope sequences and related blast searches against the Uniprot human complete proteome. BEPPE can be employed for the study of the biophysical processes at the basis of the immune recognition, as well as for immunological purposes such as the rational design of biomarkers and targets for diagnostics, therapeutics, and vaccine discovery.

Unraveling the molecular basis of immune recognition still represents a challenging task for current biological sciences, both in terms of theoretical knowledge and practical implications. Here, we describe the physical-chemistry methods and computational protocols for the prediction of antibody-binding epitopes and MHC-II loaded epitopes, starting from the atomic coordinates of antigenic proteins (PDB file). These concepts are the base of the Web tool BEPPE (Binding Epitope Prediction from Protein Energetics), a free service that returns a list of putative epitope sequences and related blast searches against the Uniprot human complete proteome. BEPPE can be employed for the study of the biophysical processes at the basis of the immune recognition, as well as for immunological purposes such as the rational design of biomarkers and targets for diagnostics, therapeutics, and vaccine discovery.