Fig. 3
Structural and functional analyses of Variants of Unknown Significance (VUS). a–c Structures of MAX homodimer and C-MYC-MAX and MXD1-MAX heterodimers in complex with DNA. a MAX-MAX homodimer crystal structure (PDB id: 1AN2) in which the subunit A (yellow for carbon atoms) and B (cyan for carbon atoms) are represented and the side chains of several invariant residues are depicted with stick models and labeled. b Crystal structure of C-MYC/-MAX heterodimer in complex with DNA (PDB id: 1NKP). MAX and C-MYC carbon atoms are represented in yellow and purple, respectively. c Crystal structure of MAX-MXD1 heterodimer in complex with DNA (PDB id: 1NLW). MAX and MXD1 carbon atoms are represented in yellow and green, respectively. In all structures presented, the MAX p.R60Q mutation is shown in magenta. Dashed lines (black and magenta) represent hydrogen bonds. The sugar-phosphate backbone of DNA is shown in orange with two selected nucleotides from each subunit shown as stick models. d MAXR60Q mutant heterodimerizes with C-MYC and MXD1 and binds to DNA. The indicated proteins were transcribed and translated in vitro and incubated with an E-box containing probe. Specific proteins/DNA complex bands are indicated on the left. Non-specific (ns) binding products present in the probe-only and vector control lanes are indicated on the left. e, f Structures of wild-type BRAF and BRAF p.K483E mutant. e Model of the BRAF kinase domain in complex with ATP (black for carbon atoms) and a Mg2+ ion (dark green), in which the side chains of five essential residues in BRAF, are shown, and labeled. The helix αC in its active conformation (dark violet) (PDB id: 4MNE) and in inactive conformation (light gray) (PDB id: 4WO5) is represented as cartoon and the side chain of the invariant E501 is depicted with stick models in two orientations. f Model of the BRAF kinase domain in which K483 is replaced by E (magenta for carbon atoms). g Proteins levels and phosphorylation level of ERK1/2 upon transient transfection of the indicated BRAF proteins in HEK 293 T cells