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. 2015 Jan 27;290(12):7707–7721. doi: 10.1074/jbc.M114.632786

FIGURE 6.

FIGURE 6.

Solution NMR studies of ARNT·TACC3 complexes are consistent with the crystal structures. a, ARNT PAS-B·TACC3 interaction observed by solution NMR. 225 μm TACC3(585–631) WT (monomeric concentration) was mixed into 100 μm 15N-labeled ARNT PAS-B. The peak broadening ratio of ARNT PAS-B residues caused by addition of TACC3 is plotted in a bar graph (mean intensity ratio, red line; mean intensity ratio −σ, red dashed line). b, ARNT PAS-B peak intensity ratio mapped onto the ARNT PAS-B crystal structure (PDB code 4EQ1 (17)) and PRE broadening effects mapped from low (blue) to high (red), with the largest broadening effects shown on the β-sheet. c, HADDOCK model of ARNT·TACC3 complex using the most severely broadened residues (peak intensity ratio ≤) as active residues. The top scoring HADDOCK model (lowest energy) is shown in yellow, superimposed on the ARNT PAS-B E362R·TACC3(585–631) crystal structure (cyan) (Cα r.m.s.d. = 2.1 Å). d, PRE effects of TACC3(585–631) Q609C-MTSL on 15N-labeled ARNT PAS-B mapped on the ARNT PAS-B·GCN4-TACC3-CT D622A/E629A crystal structure (approximate location of the MTSL group shown as red sticks). ARNT PAS-B residues with pronounced PRE effects are highlighted as spheres and colored from lower (blue) to higher (red) effects. Note that residues with the large PRE effects (red spheres) are mostly localized near the spin label. e, PRE experiments with TACC3 constructs with different spin label sites demonstrated consistent ARNT·TACC3 orientations in solution and solid state. Upper panel, individual spin label positions on TACC3 (Ser-602, Met-605, and Gln-609, shown as red sticks) and their corresponding peak broadening effects mapped on 15N-labeled ARNT PAS-B (blue, less broadening; red, more broadening). TACC3 Q609C-MTSL induced the most substantial peak broadening effects, whereas TACC3 M605C- and S602C-MTSL induced less broadening consistent with further distance from ARNT PAS-B. Lower panel, peak broadening ratio distribution induced by the corresponding spin labels. Histograms of these ratios show mean peak intensity ratio histograms shift to a lower number (to the left, more broadening) when moving the spin label toward the C terminus of TACC3, demonstrating that the average distance between spin labels and ARNT PAS-B sites (r) follows the progression rS602C > rM605C > rQ609C.