<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

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

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

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

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

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

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

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

Selected point mutations of AhR and ARNT decrease PAS-A and/or PAS-B DNA-binding ability of ligand-activated AhR/ARNT dimer.

<p>The indicated AhR and ARNT mutant constructs were synthesized <i>in vitro</i>, transformed in the presence of <i>wild-type</i> dimerization partner and 20 nM TCDD (or solvent control DMSO), and analyzed by gel retardation analysis for DNA binding. (A) Representative gel is shown as an example of the effect of selected PASB ARNT mutations on ligand-dependent AhR:ARNT DNA binding. D, DMSO; T, TCDD. (B) and (C) Protein-DNA complexes in gels as in A were quantitated and the values corresponding to the intensity of the specific complex are expressed as mean +/- standard deviation of three replicate reactions. Those values statistically different from the <i>wild-type</i>/TCDD control at <i>p</i> < 0.05 (Student’s t-test) are indicated with an asterisk (*). All experiments were performed twice and the results were consistent between the runs.</p