Fig. 4: RAS, BRAF RBD, and 14-3-3 interactions.
From: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding
a Superimposition of KRAS (from KRAS:CRAF_RBD structure PDB ID: 6XI7, colored in green) onto the autoinhibited BRAF:14-3-32:MEK complex is shown to demonstrate the fit of KRAS into the “RAS pocket” for RAS:RBD binding. b Electrostatic surface representation of the RAS:RBD-binding interface (yellow circle), with critical ionic bond-forming residues indicated on the surfaces of KRAS (PDB ID: 6XI7, left) and the RBD of the BRAF:14-3-32:MEK complex (right). c Binding affinities of the BRAF:14-3-32:MEK (top) and BRAF:14-3-32 (bottom) complexes to KRAS(GppNHp) were determined in fluorescence polarization assays. Data points present duplicate wells. The reported KD is the mean of three independent experiments. d Purified BRAF:14-3-32:MEK and BRAF:14-3-32 complexes were evaluated for their ability to bind Halo-tagged KRASG12V in pull-down assays. Blots are representative of two independent experiments with similar results. e WT or BRAF full-length proteins containing mutations in key basic RBD residues mediating ionic bond interactions with RAS were examined for binding to Halo-KRASG12V in pull-down assays. Blots are representative of three independent experiments with similar results. f Superimposition of KRAS onto the autoinhibited BRAF:14-3-32:MEK complex indicates potential steric clash and electrostatic repulsion between RAS and the 14-3-3 protomer at the RBD:14-3-3 interface upon full RAS:RBD contact. The steric clash (top insets) would occur between RAS α1-helix, switch I (SI) and β2 sheet (ribbon and surface in green) and the α8- and α9- helices of 14-3-3 (ribbon in gold). In the same region, similarly charged residues in KRAS, D30, and E31, and 14-3-3, D197, and E198, would cause electrostatic repulsion (bottom inset, RAS ribbon structure in green superimposed onto the electrostatic surface representation of the RBD and 14-3-3). Source data are provided as a Source Data file.
