Fig. 9. Structural and dynamic characterization of compounds 7 and 8 binding to MITF.
a 1H,13C-HMQC NMR spectra of apo MITF (black) and in the presence of 7 (blue) or 8 (red). Note that for 7 (but not for 8), the signals for L257δ1 and L257δ2 are weaker or absent. b Overlay of crystal structures of MITF in complex with compound 7 (PDB ID: 9H7T, ligand in blue, orientation 2) and compound 8 (PDB ID: 9H7S, ligand in orange, orientation 1). The kinked helix is shown in green and the straight helix in cyan. The additional helix, present in the crystal structures and not used in the MD simulation, is shown in purple. c, d Ligand interactions diagram for MITF(217-296) in complex with 7 and 8, respectively. In panel b the chain A, B and C labeled in the diagrams are reported in green, cyan and purple. The hydrogen bonds are shown as purple arrows. e Number of swaps per trace was calculated as in the Methods, including simulations with and without DNA as the presence or absence of DNA was not found to affect the number of calculated swaps (Supplementary Fig 9a). The “no ligand” condition has n = 5 replicates. Each compound and orientation combination plotted consists of n = 10 replicates, 5 each from simulations with and without DNA, respectively. All box plots shown use the R ggplot geom barplot() defaults, showing the median, two hinges calculated at the 25th and 75th percentiles, and two whiskers that extend from the hinges at most 1.5*(interquartile range of the relevant hinge). All data outside the whiskers are shown as individual dots. The presence of compound 7 significantly decreases the number of observed swaps, whereas presence of 8 only does so in the non-crystallographic pose of orientation 2. Significance was calculated using the two-sided Wilcoxon rank-sum test. Non-significant comparisons are not shown. Multiple comparisons were corrected for using the Benjamini-Hochberg method. ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Exact, post-correction p values for comparisons with the “no ligand” condition are provided here: (compound 7, orientation 1): p = 0.003. (compound 7, orientation 2): p = 0.00098. (compound 8, orientation 1): p = 0.971 (compound 8, orientation 2): p = 0.031. Source data for panel e are provided as a Source Data file.