Figures and data
Type II αC-in inhibitors drive BRAF dimerization through asymmetric allosteric coupling.
a, Schematic of the intermolecular FRET sensor used to quantify BRAF dimerization. b, Representative intermolecular FRET experiments measuring BRAF dimerization by type I αC-in inhibitor GDC0879 as a function of BRAF concentration. Dashed lines represent the global fit of these data to the model shown in panel c. Data for all αC-in inhibitors are shown in Supplementary Figure 3. c, Model of inhibitor-induced BRAF dimerization used for the global fitting of FRET data in panel b. B represents apo/unbound BRAF monomer, BD drug/inhibitor-bound monomer, BB apo/unbound dimer, BBD dimeric BRAF with one bound inhibitor molecule, and BBDD dimeric BRAF with two inhibitor molecules bound. These biochemical species are linked by the equilibrium dissociation constants described in the main text and methods. d,e, Equilibrium dissociation constants for dimerization (panel d) and inhibitor binding (panel e) determined from global fitting analysis of the GDC0879 experiments shown in panel b. Allosteric coupling factors α and β describe the coupling of BRAF dimerization to the first and second inhibitor binding events, respectively (see Methods), and are similar for this type I inhibitor. Dissociation constants for all inhibitors are shown in Supplementary Figure 5. f, Allosteric coupling factors α and β, as well as their product αβ, are shown for all RAF inhibitors. Error bars represent the mean ± s.e.m.; n≥3 independent experiments, each performed in duplicate. g, plots of the allosteric coupling factor ratio α/β for all αC-in inhibitors. h, Representative intermolecular FRET experiments measuring disruption of BRAFE586K dimerization by αC-out inhibitor at increasing BRAF concentrations. Dashed lines represent the global fit to the thermodynamic model shown in panel c.
Allosteric asymmetry is the driving force for paradoxical activation by type ll αC-in inhibitors.
a, Representative BRAF kinase activity data (circles, left y-axis) and induction of partially occupied BBD dimers (dashed line, right y-axis), for type II inhibitors LY3009120 and tovorafenib (left) and the type I inhibitor GDC0879 (right). Activity data represent mean values ± s.e.m.; n=3 independent experiments each performed in duplicate. Activity data for other inhibitors are shown in Supplementary Figure 10. BBD induction curves were simulated from the allosteric models parameterized with FRET data. The thickness of the band represents the 95% CI of the best-fit model from n=3 independently parameterized models. Simulations for other inhibitors are shown in Supplementary Figure 10. b, Induction landscape where the predicted amplitude of BBD induction is plotted over a wide range of α and β values. Simulations were performed using an allosteric model where KDdimer and KDdrug were kept constant and α and β were systematically varied. Inhibitors are shown mapped onto the landscape (black symbols) based on their experimentally determined α and β factors. az, AZ628, bel, belvarafenib, dab, dabrafenib, enc, encorafenib, gdc, GDC0879, ly, LY30019120, L7, L779450, pon, ponatinib, sor, sorafenib, sb, SB590885, tov, tovorafenib, tak, TAK632, vem, vemurafenib, zm, ZM33637. c, The simulated peak induction is shown as a function of coupling asymmetry α/β at two fixed values of the total coupling strength αβ, d, Simulated BBD induction magnitudes versus allosteric coupling ratios (α/β, for αC-in type I (yellow) and αC-in type II (purple) inhibitors. Data represent the mean ± s.e.m.; n≥3 independent experiments each performed in duplicate. The dashed line represents a hyperbolic fit to the data. e, Amplitude of BRAF kinase activation measured in vitro as a function of the simulated peak BBD induction for each inhibitor. Kinase activity data represent the mean ± s.e.m.; n=3 independent experiments each performed in duplicate. The slope of the linear fit, corresponding to the catalytic activity of BBD dimers, is indicated.
Type I and type II inhibitors induce distinct αC-helix conformations.
a, X-ray structures of BRAF in the apo state3, bound to the type I inhibitor GDC087941, and bound to the type II inhibitor AZ62826 (PDB IDs: 6PP9, 4MNF, 4RZW), highlighting the different αC-helix conformations stabilized by each inhibitor. Structures were aligned on the C-terminal lobe. b, (Left) Schematic of the labeling strategy used to track the conformation of the αC-helix with DEER. (Right) DEER waveforms and Gaussian distance distributions for apo BRAF (grey), BRAF bound to type I inhibitor GDC0879 (yellow), and BRAF bound to the type II inhibitors TAK632, AZ628, and LY3009120 (purple). c, Representative flow-cytometry plots showing gating used to define maximally activated (pMEKhi/pERKhi) SK-MEL-2 cells in the absence (top) and presence (bottom) of AZ628 at an activating concentration of 100 nM. White labels indicate the % of live, single cells within the maximally activated gate. d, Induction and inhibition of RAF-mediated MAPK phosphorylation in SK-MEL-2 cells treated with the type II inhibitor AZ628 (left, purple) and the type I inhibitor GDC0879 (right, yellow). The overlaid dashed lines represent the inhibitor affinities for the first and second binding sites on the dimer, defined by 
The αC-helix in the BRAF dimer dynamically samples multiple conformational states.
a, 19F NMR spectrum of apo BRAF labeled on the αC-helix (Q493C) with BTFA. The raw spectrum (grey line) was fit to a multi-component Lorentzian model (dotted line). The deconvoluted spectrum consists of three unique resonances that correspond to one αC-out state (red) and two αC-in states (blue and yellow). Resonance assignments are shown in Supplementary Figures 14a and 14c. b, Representative 19F NMR T2 relaxation profiles showing the peak areas of individual deconvoluted peaks shown in panel a as a function of T2 decay time. T2 decay parameters for each component were extracted by single-exponential fits. The inset shows a comparison of these T2 values with T2* values calculated from the spectral linewidths of each component via the relationship T2*=1/(π x linewidth). Data represent the mean ± s.e.m.; n=4 independent experiments. c, Variable-temperature 19F NMR experiments. Spectra collected at lowest (light blue) to highest (dark blue) temperatures are shown. The deconvoluted dimer peaks are also shown as dotted lines for the lowest temperature. The inset shows the relative peak areas of αC-innarrow (yellow) and αC-inbroad (blue) peaks as a function of temperature. Data represent the mean ± s.e.m.; n=3 independent experiments. d, Schematic representation of our model for how dynamic heterogeneity in the BRAF dimer contributes to paradoxical activation. The relatively static αC-innarrow (yellow) and more dynamic αC-inbroad (blue) states observed by 19F NMR experiments are highlighted in the context of drug binding to monomers and dimers, with the biochemical species arranged in the same manner as in Figure 1c. This model can be further visualized with a free-energy diagram showing the αC-innarrow and αC-inbroad states similarly populated and in slow exchange. The αC-inbroad state consists of multiple conformational states separated by small energy barriers resulting in exchange on the intermediate time scale.
