The yin–yang of kinase activation and unfolding explains the peculiarity of Val600 in the activation segment of BRAF
- Centre for Genomic Regulation, Spain
- Universitat Pompeu Fabra, Spain
- Barcelona Institute of Science and Technology, Spain
- Institució Catalana de Recerca i Estudis Avançats, Spain
Figures
Figure 1 with 3 supplements
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 …
Figure 1—figure supplement 1
BRAF activation cycle.
The 14-3-3 dimer binds to Ser365 at the N-terminus and to Ser729 at the C-terminus, maintaining the kinase in a closed, inactive conformation. Dephosphorylation of the N-terminal phospho-Ser365 by …
Figure 1—figure supplement 2
Cancer mutation frequencies in the hydrophobic pocket of BRAF.
Each position in the hydrophobic pocket region is shown (rows) and mutation frequencies for the respective mutations (columns). The mutation frequencies are colored according to the absolute number …
Figure 1—figure supplement 3
Basic principles of the FoldX force field, FoldX-based modeling, and the application of structure-energy calculations on mutations in BRAF’s hydrophobic pocket.
(A) Basic scheme of a folded and unfolded kinase and the associated folding energy (△G). (B) List of intramolecular forces contributing and opposing folding, which is integrated into the FoldX force …
Figure 2
AS loop residues in 28 BRAF kinase structures and comparison with B-factors.
(A) Percentage of the 28 BRAF X-ray structures that have a given AS residue solved. (B) Percentage of presence of AS loop amino acids in the X-ray structures, mapped onto a BRAF ribbon diagram (see …
Figure 3 with 4 supplements
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…
Figure 3—figure supplement 1
Mutations causing destabilization of the inactive loop and comparison with cancer frequencies.
(A) Mutations having destabilization of the inactive loop above the threshold (‘loop energy’), sorted by decreasing energy value. Colors indicate the number of cancer mutations. (B) Comparison of …
Figure 3—figure supplement 2
Mutations causing destabilization of the inactive structure above the threshold.
Colors indicate the number of cancer mutations.
Figure 3—figure supplement 3
Additional Western blots supporting Figure 3B.
Western blots of biological replicates of BRAF WT, V600E, V600D, V600K, V600M, V600A, V600G, and V600W used for the quantifications shown in the bar diagram of Figure 3B.
Figure 3—figure supplement 4
Additional Western blots supporting Figure 3C.
(A) Western blots of technical replicates of BRAF WT, V600E, V600H, and L597Y used for the quantifications shown in the bar diagram of Figure 3C. (B) Additional biological replicates for all BRAF …
Figure 4 with 6 supplements
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 …
Figure 4—figure supplement 1
Original western blots of spliced out lanes shown in Figure 4C and D.
(A) Original Western blot and experimental procedure supporting Figure 4C. Supernatant (‘SUP’) or pellet (‘PELLET’) fractions of BRAF WT and mutants V600E, V487E, V600W, and L525E were each run on …
Figure 4—figure supplement 2
Comparing experimental protein solubility with FoldX predicted folding energies.
Comparing the ratios of BRAF expressed in the soluble and insoluble fractions with the FoldX folding energies. The correlation coefficient is 0.67.
Figure 4—figure supplement 3
MEK phosphorylation of wild-type and V600E, V487E, and L525E mutant BRAF in the supernatant.
(A) Western blot analysis of MEK-phosphorylation after expression for 24 hr in normal medium and ImageJ quantification (using two biological replicates). (B) MEK-phosphorylation levels normalized by …
Figure 4—figure supplement 4
Conformations of Lys601 found in all structures having position 601 solved, and an overlay of ten active-like BRAF structures.
(A) Close-up of the 4MNE structure indicating the salt bridge between Arg575 and Glu611 that is conserved in all Raf kinases. Lys601 points in the direction of this salt bridge. (B) Superimposition …
Figure 4—figure supplement 5
Biological replicates in minimal (serum-free) growth medium.
https://doi.org/10.7554/eLife.12814.018
Figure 4—figure supplement 6
Analysis of the interactions in the BRAF RD motif, and expression levels of BRAF wild-type and the single V600E, E611A, and double V600E/E6111A mutants.
(A) Illustration of salt bridges that are proposed to stabilize the active conformation. The structural representation was done with the SwissPdbViewer, using PDB entry 4MNE. The bottom panel shows …
Figure 5 with 1 supplement
Quantitative contribution of individual factors to the prediction of cancer frequencies.
(A) Comparison of real and predicted cancer frequencies (labelled ‘real value’ and ‘predicted value’) for one exemplary random forest prediction (run 16). Black dots represent mutations that were in …
Figure 5—figure supplement 1
Random forest analyses without V600E in the training set.
(A) Results from the random forest analyses without V600E in the training set. Abbreviation for parameters: 1, destabilization of inactive conformation/folding; 2, destabilization of active …
Figure 6
Schematic diagram depicting the relationship between structural flexibility, destabilization of the hydrophobic pocket, and cancer frequencies.
The effect of a mutation on folding depends on the structural flexibility of the respective hydrophobic pocket where the mutated amino acid is located. In a region with higher structural flexibility …
Additional files
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Supplementary file 1
Summary of structure-energy and statistical properties of mutations in the hydrophobic pocket of BRAF and parameters used for random forest analyses.
This table summarizes on sheet 1 for all mutations in the hydrophobic pocket the mutation frequencies in cancer, the average energies predicted using FoldX using active or inactive BRAF template structures, the respective B-factors, the presence of amino acid residues in solved BRAF X-ray structures, the nucleotide substitution frequency, the hydrophobic solvation energy, the change in codon usage, and the energetic contribution of a salt bridge predicted to stabilize the active conformation. Sheet 2 summarizes for all mutations in the hydrophobic pocket the values used to for the seven parameters evaluated in the random forest analyses.
- https://doi.org/10.7554/eLife.12814.023
