Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors
- Department of Pharmacology, University of Minnesota, United States
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, United States
- Department of Chemistry, University of Minnesota, United States
- Center for Immunology, University of Minnesota, United States
- Masonic Cancer Center, University of Minnesota, United States
Figures
Figure 1 with 11 supplements
Type II αC-in inhibitors drive BRAF dimerization through asymmetric allosteric coupling.
(a) Schematic of the intermolecular FRET sensor used to quantify BRAF dimerization. A detailed structrual model of the FRET sensor is shown in Figure 1—figure supplement 1. (b) Representative …
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Figure 1—source data 1
Tabulated source data for Figure 1.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-data1-v1.zip
Figure 1—figure supplement 1
X-ray structures showing the BRAF dimer labeled with fluorophores and structural features of type I and type II inhibitor binding modes.
(a) X-ray structure (PDB: 5C9C) showing the BRAF dimer covalently labeled at K547 with Alexa Fluor 488 C5 maleimide using mtsslSuite (http://www.mtsslsuite.isb.ukbonn.de/). (b) Stopped flow …
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Figure 1—figure supplement 1—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Quantifying apo BRAF dimerization affinity (KDdimer) and dimer induction by GDC0879.
(a) Intermolecular FRET experiments tracking BRAF dimerization as a function of GDC0879 concentration were carried out at high BRAF concentrations in order to define the relatively weak apo BRAF …
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Figure 1—figure supplement 2—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp2-data1-v1.zip
Figure 1—figure supplement 3
Intermolecular FRET experiments measuring inhibitor-induced BRAF dimerization.
Representative data for all αC-in inhibitors showing BRAF dimerization for type I (yellow) and type II (purple) inhibitors. Gradients from light to dark represent experiments done at increasing BRAF …
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Figure 1—figure supplement 3—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp3-data1-v1.zip
Figure 1—figure supplement 4
One- and two-dimensional error surfaces show that global fitting yields well-constrained parameters for the allosteric model.
Representative one-dimensional error surface analyses of the global fit parameters from FRET experiments with the αC-in type II inhibitor belvarafenib (a) and the αC-out inhibitor dabrafenib (b). …
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Figure 1—figure supplement 4—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp4-data1-v1.zip
Figure 1—figure supplement 5
Equilibrium dissociation constants derived from global fitting of FRET data for all type II inhibitors.
Dissociation constants for BRAF dimerization (blue) and inhibitor binding affinity (red) derived from the global fitting analysis of the FRET dimerization data. Allosteric coupling factors α and β …
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Figure 1—figure supplement 5—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp5-data1-v1.zip
Figure 1—figure supplement 6
Equilibrium dissociation constants derived from global fitting of FRET data for type I αC-in and αC-out inhibitors.
Dissociation constants for BRAF dimerization (blue) and inhibitor binding affinity (red) derived from the global fitting analysis of the FRET dimerization data. Allosteric coupling factors α and β …
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Figure 1—figure supplement 6—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp6-data1-v1.zip
Figure 1—figure supplement 7
Inhibitor affinities for monomeric BRAF (KDdrug) measured by intramolecular FRET.
(a) Schematic highlighting the equilibrium dissociation constant KDdrug that is being independently measured in the context of the thermodynamic model used for global fitting. (b) Schematic showing …
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Figure 1—figure supplement 7—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp7-data1-v1.zip
Figure 1—figure supplement 8
The A481F active site mutation blocks inhibitor binding and prevents inhibitor-induced dimerization.
(a) A schematic demonstrating how the A481F active site mutation eliminates the effects of β on BRAF dimerization by blocking inhibitor binding and preventing the formation of BRAF dimers with both …
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Figure 1—figure supplement 8—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp8-data1-v1.zip
Figure 1—figure supplement 9
The binding of a single αC-in type II molecule to the BRAF dimer is sufficient to dramatically increase dimerization affinity.
(a) Representative intermolecular FRET dimerization data showing the formation of BRAFA481F/BRAF heterodimers (orange) and BRAF/BRAF homodimers (blue) as a function of inhibitor concentration. The …
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Figure 1—figure supplement 9—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp9-data1-v1.zip
Figure 1—figure supplement 10
Intermolecular FRET experiments measuring the disruption of BRAFE586K dimerization by αC-out inhibitors.
Representative data showing BRAFE586K dimerization as a function of αC-out inhibitors (red) and the αC-in inhibitor LY3009120 (blue) for comparison. Gradients from light to dark represent …
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Figure 1—figure supplement 10—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp10-data1-v1.zip
Figure 1—figure supplement 11
Only asymmetric allosteric models correspond to realistic levels of catalytic activity of BBD dimers for type II inhibitors.
(a) For some datasets, removing constraints on the BRAF concentrations used in the global fitting analysis resulted in two possible solutions, as shown for a representative case of fitting FRET data …
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Figure 1—figure supplement 11—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig1-figsupp11-data1-v1.zip
Figure 2 with 2 supplements
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 …
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Figure 2—source data 1
Tabulated source data for Figure 2.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig2-data1-v1.zip
Figure 2—figure supplement 1
Simulations of partially occupied BRAF dimer formation and in vitro kinase activity.
The formation of partially occupied ‘BBD’ BRAF dimers was simulated as a function of inhibitor concentration using the allosteric model (Figure 1c) parameterized for each inhibitor via the global …
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Figure 2—figure supplement 1—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
BBD induction amplitude and peak catalytic turnover.
(a) Peak BBD induction amplitudes from individual simulations shown in Figure 2—figure supplement 1 are shown for type I (yellow) and type II (purple) αC-in inhibitors. Data represent the mean ± …
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Figure 2—figure supplement 2—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig2-figsupp2-data1-v1.zip
Figure 3 with 3 supplements
Type I and type II inhibitors induce distinct αC-helix conformations and promote MAPK/ERK pathway activation in SK-MEL-2 cells.
(a) X-ray structures of BRAF in the apo state (Park et al., 2019), bound to the type I inhibitor GDC0879 (Haling et al., 2014), and bound to the type II inhibitor AZ628 (Karoulia et al., 2016) (PDB …
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Figure 3—source data 1
Tabulated source data for Figure 3.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig3-data1-v1.zip
Figure 3—figure supplement 1
Measuring the conformation of the αC-helix with double electron-electron resonance (DEER).
(a) X-ray structures of BRAF in the apo state (gray) (PDB ID: 6PP9), bound to type I inhibitors (yellow) (PDB IDs: 4MNF, 2FB8) and type II inhibitors (purple) (PDB IDs: 4RZW, 6P3D, 5C9C, 6XFP, 4KSP, …
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Figure 3—figure supplement 1—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
Inhibitor-induced MAPK/ERK pathway activation in SK-MEL-2 cells.
Paradoxical activation of MAPK/ERK signaling in SK-MEL-2 cells by type I (yellow) and type II (purple) RAF inhibitors measured by flow cytometry. The dashed lines represent the inhibitor affinities …
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Figure 3—figure supplement 2—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig3-figsupp2-data1-v1.zip
Figure 3—figure supplement 3
Western blot confirming the presence of all RAF isoforms in SK-MEL-2 cells.
SK-MEL-2 cells were treated with the indicated amount of type II inhibitor AZ628 or type I inhibitor GDC0879 for 1 hr. Cell lysates were immunoblotted for all endogenous RAF isoforms. Note that …
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Figure 3—figure supplement 3—source data 1
Uneditited western blots.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig3-figsupp3-data1-v1.zip
Figure 4 with 2 supplements
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 3-bromo-1,1,1-trifluoroacetone (BTFA). The raw spectrum (gray line) was fit to a multi-component Lorentzian model (dotted line). …
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Figure 4—source data 1
Tabulated source data for Figure 4.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig4-data1-v1.zip
Figure 4—figure supplement 1
19F NMR resonance assignments and experiments confirming the presence of dynamic heterogeneity within the BRAF dimer.
(a) 19F NMR spectra of BRAF labeled on the αC-helix with 3-bromo-1,1,1-trifluoroacetone (BTFA) in the presence of ATP. ATP causes an increase in the upfield resonance and decrease in the downfield …
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Figure 4—figure supplement 1—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 2
Validation of BRAF labeling, stability, and purity.
(a) Mass spectra of BRAF16mut (theoretical molecular weight: 32,204 Da), BRAFDB+Q493C+Q664C labeled with two 4-maleimido-TEMPO spin probes (theoretical molecular weight: 32,638 Da), BRAFQ493C …
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Figure 4—figure supplement 2—source data 1
Tabulated source data.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Raw gel from panel c.
- https://cdn.elifesciences.org/articles/95481/elife-95481-fig4-figsupp2-data2-v1.zip
Additional files
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Supplementary file 1
Inhibitor sources and validation by mass spectrometry.
- https://cdn.elifesciences.org/articles/95481/elife-95481-supp1-v1.docx
