Fig. 3: Analysis of the RBD:14-3-3 interface.
From: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding
a Region of RBD:14-3-3 contact, with RBD and 14-3-3 residues at the contact interface depicted as light gray and dark gray, respectively. Interacting residues are labeled (top). Electrostatic surface representation of the RBD and 14-3-3, with blue and red representing positively and negatively charged areas, respectively. Regions of contact between the RBD and 14-3-3 lay within the yellow circles (bottom). b The 14-3-3 α8-helix, α9-helix, and the connecting loop 8 contact the α1-helix and loop 3 of the BRAF RBD. The RBD is colored in orange, 14-3-3 in gold and the interacting residues are labeled (top). Sequence alignment of RBD residues in the α1-helix and loop 3 of human BRAF, CRAF, and ARAF (bottom). Residues at the RBD:14-3-3 interface are denoted by the shaded gray box, and identically conserved residues are shown in red. Symbols under the alignment denote the degree of conservation as follows: (*) indicates positions that have a fully conserved residue, (:) indicates conservation between groups with strongly similar properties, and (.) indicates conservation between groups with weakly similar properties. c NanoBRET assay monitoring the interaction between WT or mutant BRAFREG-Halo proteins and Nano-BRAFKD in live cells to determine the effect of the indicated mutations on the ability of BRAF to form a stable, autoinhibited complex. BRET signals (normalized to WT set at 100) of quadruplicate wells from five independent experiments were used to generate a box and whiskers plot, with the center line representing the median, the box limits extending from the 25th to 75th percentile, and the whiskers extending from the minimum to maximum values. d Lysates of 293FT cells transiently expressing Nano-WT-BRAFKD alone or co-expressing Nano-WT-BRAFKD with increasing amounts of WT-, M186A/M187A-, or M186E/M187E-BRAFREG-Halo were examined by immunoblot analysis for WT-BRAFREG-Halo, Nano-WT-BRAFREG, and pMEK levels. Blots are representative of three independent experiments with similar results. e NIH-3T3 cells were infected with retroviruses encoding the indicated WT or mutant FLAG-BRAFFL proteins. Three weeks post-infection, foci were visualized by methylene blue staining. Stained plates are representative of three independent experiments with similar results. Source data are provided as a Source Data file.
