Overall representation of the PASA.4F3L and PASB.4F3L and related mutations that decrease DNA binding activity.

<p>AhR is colored in green, ARNT in cyan, the PAS dimerization interfaces in orange, the positions of the mutated residues in yellow, the TCDD ligand is represented in dark grey spheres. (A) The PAS dimer models are superimposed to the full-length 3D scaffold of the CLOCK:BMAL1 template; the orientation of the bHLH motif (highlighted in grey) is taken from the experimental atomic coordinates of the 4F3L crystallographic structure. (B) Mapping of the mutations along the <i>PASB</i>.<i>4F3L</i> dimer model. The coordinates of the <i>wild-type</i> residues are highlighted in yellow, putative rotamers of the mutated residues are shown in grey sticks. (C) Mapping of the mutations along the <i>PASA</i>.<i>4F3L</i> dimer model.</p

The four PAS dimer models proposed.

<p>The AhR domains are colored in green, the ARNT ones in cyan. Residues defining the protein-protein interface (according to PISA analysis) are highlighted in yellow and residues defining the specific ∆<i>G signatures</i> (according to the Rank Products approach) are highlighted in magenta. For the PAS-B dimers, the AhR bound ligand TCDD is depicted in spheres.</p

Rank Products profiles from the comparison of the PAS-A and PAS-B dimer models.

<p>Asterisks denotes residues with a significant LOG(<i>RP</i>) value (<i>e-value</i> ≤ 0.05 after 10,000 random permutations). (A) Residues with a negative LOG(<i>RP</i>) value define the <i>PASA</i>.<i>4F3L</i> ∆<i>G signature</i>; residues with a positive LOG(<i>RP</i>) value define the <i>PASA</i>.<i>4M4X</i> ∆<i>G signature</i>. (B) Residues with a negative LOG(<i>RP</i>) value define the <i>PASB</i>.<i>4F3L</i> ∆<i>G signature</i>; residues with a positive LOG(<i>RP</i>) value define the <i>PASB</i>.<i>3F1P</i> ∆<i>G signature</i>.</p

X-ray structures of the templates adopted for homology modeling.

<p>The PAS-A domains are colored in green and blue, the PAS-B domains in orange and violet; the residues involved in the dimerization interfaces are highlighted in yellow. (A) Murine CLOCK:BMAL1 complex, N-terminal region. (B) Human HIF2α:ARNT complex, PAS-B dimer. (C) Murine PAS-A AhR homodimer.</p

Electrostatic Potential Surface (EPS) of the dimer models.

<p>In each panel, the ARNT domain is rotated of 180 degree with respect to the AhR domain, to obtain a representation of the dimer as an “open book”. The potential range is defined in kT/e units according to the DelPhi software. The secondary structure elements mainly involved in the PPI definition are highlighted with green circles. (A) <i>PASA</i>.<i>4F3L</i> dimer model. (B) <i>PASA</i>.<i>4M4X</i> dimer model. (C) <i>PASB</i>.<i>4F3L</i> dimer model. (D) <i>PASB</i>.<i>3F1P</i> dimer model.</p

<i>Hot spot</i> list from PAS-A dimer models, with related scores from the PPI prediction tools herein adopted.

<p><i>Hot spot</i> list from PAS-A dimer models, with related scores from the PPI prediction tools herein adopted.</p

Residue contacts for the dimer models derived from the PPI prediction methods and visual inspection.

<p>Residues involved in electrostatic non-bonded interactions (i.e. H-bonds and/or salt bridges) are highlighted with yellow spheres. In both the AhR and ARNT domains, helices are shown in cyan, strands in purple. (A) <i>PASA</i>.<i>4F3L</i> dimer model. (B) <i>PASA</i>.<i>4M4X</i> dimer model. (C) and (D) PASB.4F3L dimer model. (E) PASB.3F1P dimer model.</p

Overall evaluation of the dimerization interfaces of the models and the templates adopted (in brackets).

<p>Overall evaluation of the dimerization interfaces of the models and the templates adopted (in brackets).</p

Electrostatic Potential Surface (EPS) of the dimer models.

<p>In each panel, the ARNT domain is rotated of 180 degree with respect to the AhR domain, to obtain a representation of the dimer as an “open book”. The potential range is defined in kT/e units according to the DelPhi software. The secondary structure elements mainly involved in the PPI definition are highlighted with green circles. (A) <i>PASA</i>.<i>4F3L</i> dimer model. (B) <i>PASA</i>.<i>4M4X</i> dimer model. (C) <i>PASB</i>.<i>4F3L</i> dimer model. (D) <i>PASB</i>.<i>3F1P</i> dimer model.</p

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

<div><p>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.</p></div