Table 1.
Data collection and refinement statistics.
Figure 1.
(A) Orthogonal views of the Max-E47 structure shown as a ribbon representation with one protomer colored as a rainbow by B-factor (10 (blue)≤B≤70 (red)) and the other protomer shown with basic region from Max in blue and the helix-loop-helix region from E47 in yellow. The inset highlights the bending of helix a1 in Max-E47 (blue-yellow) towards the symmetry axes of the dimer. The structures of MaxbHLHZ (light blue) and E47bHLH (light yellow) are shown as a reference. (B) Sequence of the Max-E47 chimera with the basic regions from Max and E47 colored as in panel (A). Sequences added during cloning are shaded in grey. The secondary structure elements are indicated above the sequence, with the disordered regions shown as dashed lines. The native residues of Max and E47 are indicated underneath and the two mutations that create the Max-E47Y and Max-E47YF cloning variants are marked with arrows. (C) Detail of the conformational changes in helix a1 induced by DNA binding. From left to right: Max-E47 (Max and E47 portions colored in purple and pink, respectively), MaxbHLHZ bound to DNA (teal), E47bHLH bound to DNA (yellow) and a superimposition of the three structures. The helical axes are indicated as grey lines beside each structure and kinks in the helices are marked with black arrows.
Figure 2.
Electron density maps of Max-E47.
Detailed view of the dimerization interface (A) and the basic region of Max-E47 (B). Composite omit electron density maps contoured at 1.5σ are shown as a white mesh. The two protomers of the Max-E47 dimer are shown as yellow and white color-coded sticks.
Figure 3.
Max-E47 is a dimer in solution.
(A) Size exclusion chromatography profile of Max-E47 over a Superdex75 column (GE Healthcare). Max-E47 elutes at a volume consistent with a dimer as reflected by the elution volumes of the molecular weight markers (albumin, 67 kDa; ovoalbumin, 43 kDa; chymotrypsinogen A, 25 kDa; and ribonuclease A, 13.7 kDa). (B) Continuous c(s) distributions obtained from sedimentation velocity data collected at 50 krpm, for Max-E47 (left) in 20 mM Tris pH 8.0, 0.1 M NaCl, 10 mM 2-mercaptoethanol and 5% (v/v) glycerol at loading concentrations of 3 (red), 22 (orange), 60 (green) and 130 (blue) mM. A major species is observed at 1.70 S representing a Max-E47 dimer, based on a best-fit molecular mass of 17.7±0.3 kDa (Mcalc monomer = 9.066 kDa) obtained for this species in the absence of glycerol. (C) Sedimentation equilibrium profiles for Max-E47 at 16.0°C plotted as a distribution of the interference fringe displacement vs. radius at equilibrium. Data were collected at 14 (orange), 21 (yellow), 28 (green) and 35 (brown) krpm and loading concentrations of 25 (left panel), 10 (center panel) and 5 mM (right panel). The solid lines show the best-fit analyses in terms of two non-interacting species, returning molecular masses of 17.6 and 31.8 kDa and indicating the presence of both Max-E47 dimers and tetramers. The corresponding residuals for these best-fit analyses are shown in the plots above. Statistically indistinguishable fits (within 90% confidence intervals) were obtained when data were modeled in terms of a mixture of non-interacting Max-E47 dimers and tetramers. In these cases corrections for the time-invariant noise were not carried out.
Figure 4.
Oligomerization of the Max-E47 dimer.
The crystal packing of Max-E47 suggests that dimers of Max-E47 (shown as orange and purple ribbon diagrams) can associate through crystallographic symmetry to form tetramers.
Figure 5.
Max-E47YF is a monodisperse dimer in solution.
(A) Sedimentation equilibrium profiles for Max-E47YF at 16.0°C plotted as a distribution of the interference fringe displacement vs. radius at equilibrium. Data were collected at 14 (orange), 21 (yellow), 28 (green) and 35 (brown) krpm and loading concentrations of 20 (left panel), 10 (center panel) and 5 µM (right panel). The solid lines show the best-fit analyses in terms of a single ideal solute with mass conservation constraints, returning a molecular mass of 18.8±0.3 kDa, and demonstrating that Max-E47YF is a monodisperse dimer (Mcalc monomer = 9.179 kDa). The corresponding residuals for this best-fit are shown in the plots above. The best-fit time-invariant noise is also shown in each plot shifted by +1.9 (left), +1.6 (center) and +0.75 (right) fringes. Attempts to fit these data in terms of a MaxE47-YF monomer-dimer self-association indicate dimerization affinities tighter than 1 nM with a 95% confidence upper limit of 30 nM. (B) Detailed view of the 2Fo-Fc electron density map (contoured at 1 σ) around Val59. (C) Detailed view of the 2Fo-Fc electron density map (contoured at 1 σ) around Val51.
Table 2.
Circular dichroism and DNA binding affinities.