Fig. 5: RBD residues at the 14-3-3 interface contribute to RAS binding.
From: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding
a WT-BRAF-Rluc and the indicated M186/M187 mutants were assessed for binding to Venus-KRASG12V in co-immunoprecipitation assays. Lysates were also monitored for BRAF-Rluc protein levels. Blots are representative of three independent experiments with similar results. b BRET saturation curves are shown examining the interaction of WT- or M186/ M187 mutant BRAF-Rluc proteins with Venus-KRASG12V in live cells. As a control, an RBD protein containing the R188L mutation that disrupts the RAF–RAS interaction was also evaluated. BRETmax and BRET50 values are listed ±standard error on the right. Saturation curves were repeated three times with similar results. c Binding of Venus-KRASG12V to WT or mutant Halo-BRAF RBD proteins was assessed in pull-down assays. Blots are representative of three independent experiments with similar results. d Modeling the effect of substituting the CRAF K87/V88 amino acids for K87E/V88E, which would generate electrostatic repulsion (indicated by dark red lines) with polar (Q25, T35, and Y40) and negatively charged (E31 and D33) KRAS residues. e Modeling the effect of substituting the CRAF K87/V88 amino acids for K87W/V88W, K87M/V88M, or K87A/V88A in the RBD:RAS structure (KRAS:CRAF_RBD_CRD structure PDB ID: 6XI7, colored in green for KRAS, orange for CRAF RBD, and violet for CRAF CRD). Gray lines indicate potential non-electrostatic interactions and blue dashed lines shows potential hydrogen bonds. Source data are provided as a Source Data file.
