Figure 1A Structure of the DNA-bound Myc/Max dimer (Nair & Burley, 2003), with an alignment of the Myc and Max basic regions (numbering based on the 439 a.a. human Myc protein (GenBank nr. AAA36340.1). B H359 and E363 in Myc establish H-bonds with the complementary G6 and C1' bases, respectively, E363 forming an additional bond with A2. R364, R366 and R367 contact the phosphodiester backbone, R367 interacting also with G4. Note that, in keeping with the symmetric configuration of the Myc/Max dimer (panel A) and with the similar structure of the Max homodimer (Ferre-D'Amare et al., 1993), the corresponding residues in Max (R33/35/36, and H28/E32) form equivalent contacts with the other half of the E-box palindrome. C Far-UV CD spectra at 20°C (top) and thermal denaturation monitored at 222 nm (bottom) for Myc (red) and Max (black) bHLH-LZ constructs (at 16 μM and 8μM, respectively), alone or in combination (Myc+Max), as indicated. SUM Myc+Max: theoretical sum of the individual Myc and Max curves. The Myc variant used (WT, RA or HEA) is indicated at the top. The increased ellipticity signals at 222 nm and increased melting temperatures of the experimental Myc+Max mixtures (blue) compared to the theoretical sums (grey) demonstrate that MycWT, MycHEA and MycRA heterodimerize with Max to comparable extents. D EMSA of the Max bHLH-LZ construct alone (at 2 μM) or in the presence of either Myc construct (MycWT, MycHEA or MycRA, all at 6 μM). The polypeptides were incubated in presence of 500 nM of the fluorescently-labeled IRD-CACGTG probe and the complexes separated on a native polyacrylamide gel, as described (Beaulieu et al., 2012). E EMSA titration experiment, with increasing concentrations of either Myc bHLH-LZ variant (WT or HEA) in presence of a fixed amount of the Max bHLH-LZ (2 μM) and of the fluorescently-labeled IRD-CACGTG probe (500 nM). The plot shows the mean and standard deviation of the quantification of the shifted Myc/Max bands as a function of the Myc concentration (the experiment was performed in triplicate). % Bound probe: fraction of the IRD-CACGTG probe associated with the Myc/Max complex. HEA/WT Kd ratio: Kd value for MycHEA/Max/CACGTG divided by the Kd value for MycWT/Max/CACGTG. The apparent Kd values were 3.78 μM for MycWT/Max and 5.57 μM for MycHEA/Max. Data information: Note that the differences in apparent Kd values between panels E and F-H can be explained by the different thresholds required to detect quantifiable complexes in the protein titration experiment (E), as opposed to a loss of signal intensity from an already detectable complex in the presence of unlabeled competitor (F-H): as such, competition experiments provide a better approximation of actual binding affinities. (F-H): The plots show the mean and standard deviation. The experiments were performed in triplicate. Unpaired Student t-test was used to compare IC50 values and expressed as p-values. F EMSA competition of the complex between Myc/Max and the fluorescently-labeled IRD-CACGTG probe with increasing concentrations of an unlabeled CACGTG probe (0.195, 0.390, 0.781, 1.563, 3.125, 6.25, 12.50 and 25 μM). The plot at the bottom shows the mean and standard deviation of the quantification of the shifted Myc/Max bands as a function of unlabeled probe concentration (the experiment was performed in triplicate). HEA/WT Kd ratio: as in E. The apparent Kd values were 387 nM for MycWT/Max and 622 nM for MycHEA/Max. G Same as F, with an unlabeled GGATCC probe as competitor. The apparent Kd values were >25 µM for MycWT/Max and 14.5 µM for MycHEA/Max. H Same as F, with an unlabeled CACGTC probe as competitor. The apparent Kd values were 674 nM for MycWT/Max and 280 nM for MycHEA/Max. Note that that last two lanes of the upper gel were loaded in inverted order, and for this reason were cropped and flipped horizontally in the final layout. Da Data information: Note that the differences in apparent Kd values between panels E and F-H can be explained by the different thresholds required to detect quantifiable complexes in the protein titration experiment (E), as opposed to a loss of signal intensity from an already detectable complex in the presence of unlabeled competitor (F-H): as such, competition experiments provide a better approximation of actual binding affinities. (F-H): The plots show the mean and standard deviation. The experiments were performed in triplicate. Unpaired Student t-test was used to compare IC50 values and expressed as p-values.