Fig 2: Structure-energy predictions and experimental analysis of mutations affecting the folding of BRAF and analysis of phosphorylation of Thr599 and Ser602 to keep the AS in a fixed active state.(A) Structural representations of the localization of Val487, Leu525, and Phe498 in BRAF (PDB entry 4EHE). (B) Destabilization of inactive and active states for V487E, L525E, and F498S BRAF (folding mutants) as predicted by FoldX. (C) Western blot analysis for BRAF mutations affecting folding. (D) Western blot analysis for BRAF F498S folding mutations. (E) Plot of BRAF soluble to insoluble ratios for the WT and mutations shown in the Western blots from pane (C) and (D), sorted in a similar order as in Figure 3B. Bar graphs show the results from two biological replicates. The soluble/insoluble value for BRAF F498S was estimated (see main text and represented with a star). (F) Illustration of the salt bridges that are proposed to stabilize the active conformation. The structural representation was done with the SwissPdbViewer, using PDB entry 4MNE. (G) Western blot analysis for the selected V600E and V600K mutations in combination with the T599A/S602A mutations expressed 24h in normal medium. (H) Quantifications of MEK phosphorylation levels normalized by total BRAF from (G) using ImageJ. Bars represent at least four biological replicates for the abundance of MEK-P normalized to total BRAF.DOI: http://dx.doi.org/10.7554/eLife.12814.013

Fig 3: TEMTAC system components.(a) Cre-LoxP mediated generation of plasmid fusions is shown in a schematic view. Acceptor A plasmid module is incubated with two Donor modules, D1 and D2 in the presence of Cre recombinase. Concomitant assembly (Cre) and excision (De-Cre) reactions occur until equilibrium is reached. Acceptor-Donor (A-D1, A-D2) and Acceptor-Donor-Donor (A-D1-D2 or “pTEMTAC”) fusion plasmids co-exist with educt plasmids when equilibrium is reached. Acceptor A contains a common origin or replication (ColE1), Donors D1 and D2 contain conditional origins of replication derived from phage R6K?, rendering their propagation in regular cloning strains dependent on productive Cre fusion with Acceptor A. (b) Donor D1 (pMDC-RNAiDual) is shown in a schematic view. This Donor provides cassettes for multiple shRNA production. (c) shRNA-mediated downregulation of SHP2, HRAS and BRAF after transfection with a Donor D1 producing specific shRNAs. Transfected HEK293 (for HRAS and BRAF) or GH-HEK293 cells were lysed and analysed by Western blotting. (d) Four Donor plasmid variants D2.1 to D2.4 are shown schematically, which realize four distinct dynamic ranges of exogenous protein expression. Abbreviations: Cre, Cre recombinase enzyme; LoxP, imperfect inverted repeat recognized by Cre; GOI, gene of interest; A-D1, fusion of Acceptor A with Donor D1; A-D2, fusion of Acceptor A with Donor D2 (or variants); A-D1-D2, complete fusion of Acceptor A with Donors D1 and D2 (or variants); shRNA, small hairpin RNA sequence; I-SceI, PI-PspI and PI-SceI are homing endonucleases; H1, U6, CMV and CAG are common mammalian active promoters; pA and SV40 are common poly-adenylation signals; TRE-pCMVmin, tetracycline response element with minimal CMV promotor; rtTA, tetracycline transactivator with (random) mutagenesis derive Tet repressor part of the transactivator gene; Sp, Cm, Hygr and Zeo denote resistance marker genes for spectinomycin, chloramphenicol, hygromycin and zeocin, respectively; YFP, yellow fluorescence protein; TetR, tet repressor gene; TetR-KRAB, tetracycline-controlled hybrid protein of TetR with the KRAB silencing domain of human Kid1; IRES, internal ribosome entry site; Frt, FLP recognition target; ColE1, common colicin E1 derived replication origin; R6K?, conditional origin derived from R6K? phage.

Fig 4: Overall structure of the kinase domain of BRAF, zoom into the hydrophobic pocket of BRAF, and active- and inactive-like BRAF kinase domain 3D structures used for structure-energy calculation.(A) Structure of the BRAF kinase, with functional regions indicated. The BRAF kinase domain has two subdomains, a small N-terminal lobe and a large C-terminal lobe. The small lobe contains the nucleotide-binding pocket and the phosphate-binding loop, while the large lobe binds the proteins substrates and contains the catalytic loop. The two lobes are spatially connected through the activation segment (AS) of the large lobe. Sequentially, the N- and C-terminal lobes are connected by the hinge, and the AS is part of the C-lobe that interacts with the N-lobe. Movement of the two lobes relative to each other opens and closes the cleft. (B) The hydrophobic pocket around amino acid Val600 represented using the backbone and side chain view. Backbone residues are colored according to their location in the protein (see Figure 1A). Specifically, Leu597, Ala598, Val600, and Trp604 of the AS together with, Phe468, Leu525, Leu485, Val487, Phe498, and Ala497 of the N-terminal subdomain build the hydrophobic pocket. All BRAF structural representations were done with SwissPdbViewer, using PDB entry 4EHE (chain B of the crystallographic unit). (C) Superimposition of active-like BRAF kinase structures. The structural representations were made using SwissPdbViewer (PDB entries 4MNE, 3OG7 and 4MNF). (D) Superimposition of inactive-like BRAF kinase structures. Structural representations were made using SwissPdbViewer (PDB entries 4EHE and 3TV6). (E) Pairwise correlation of FoldX energies for mutations in the hydrophobic pocket derived from active and inactive structures. Similar correlation results were obtained from FoldX energies using a recently published 3D structure of inactive monomeric BRAF (Thevakumaran et al. (2015); PDB entry 4WO5, which is missing four residues in the AS/ data not shown).DOI: http://dx.doi.org/10.7554/eLife.12814.003

Fig 5: Structure-energy predictions and experimental analysis of mutations in the hydrophobic pocket of BRAF.(A) Comparison of the number of cancer mutations (>0) with destabilization of the hydrophobic pocket as predicted by FoldX (average energy values of 1EHE and 3TV6, ‘FoldX ??G BRAF_inactive_loop’). (B) Representative Western blot (upper panel) for selected Val600 mutations expressed 24 hr in normal medium and quantified using ImageJ (lower panel). Two out of at least six biological replicates are shown. Bar graph shows the results of six biological replicates for the abundance of MEK-P normalized to total BRAF. (C) Representative Western blot (upper panel) analysis for selected single and triple nucleotide substitution BRAF mutations expressed 24 hr in normal medium and quantified using ImageJ (lower panel). Two out of at four biological replicates are shown. Bar graphs show the results of two biological and two technical replicates for the abundance of MEK-P normalized to total BRAF. (D) Correlation of FoldX energies with MEK phosphorylation normalized by the total BRAF levels. FoldX energies were calculated from the inactive loop energy [BRAF_inactive_loop] minus the FoldX energies derided from active structures [BRAF_active] plus the hydrophobic solvation energy as a factor in the FoldX force field [BRAF hydr_solv_energy].DOI: http://dx.doi.org/10.7554/eLife.12814.008