(Click on the small image to get a higher-resolution version.)
Figure 1 - Structure of the B-Raf:14-3-3 complex.
(A) Left, schematic diagram of the structure. Middle and right, two orthogonal views of the molecular surface of the cryo-EM model. The two B-Raf kinases in the dimer are shown in cyan and magenta, respectively, and the 14-3-3 dimer is shown in gray. B-RafIN (cyan) is positioned closer to 14-3-3 than is B-RafOUT (magenta). The schematic diagrams at the bottom of the panel denote the boundaries of the kinase domains and the C-tails of B-Raf that are included in the structural model, and the terms used to identify segments of the C-tails. The kinase domains and the C-tails are not to scale, and that is indicated by the breaks. Dashed lines indicate regions for which there is no interpretable density.
(B) A view of the B-Raf:14-3-3 complex, looking down the 2-fold symmetry axis of the B-Raf kinase dimer. The B-Raf kinase dimer seen in the cryo-EM structure closely resembles the dimeric structure of ON-state B-Raf bound to MEK (PDB ID: 4MNE) (22). Helix αG of B-RafOUT has weaker density compared to the rest of the complex, and is shown in gray.
Figure 2 - Interaction of the distal tail segment of B-RafOUT with the active site of B-RafIN.
(A) Orthogonal views of the cryo-EM structure of the B-Raf:14-3-3 complex. On the left, the C-tail of B-RafOUT (magenta) is seen bound to 14-3-3 (gray) and the distal tail segment enters the active site of B-RafIN (cyan).
(B) View of the ATP binding site of B-RafIN (cyan), with cryo-EM density shown in gray. Residues in the distal tail segment of B-RafOUT (magenta) are identified by asterisks.
(C) The hydrophobic sidechains of the C-spine of B-RafIN are shown as yellow spheres, with two sidechains of the B-RafOUT distal tail segment (magenta) completing the C-spine.
Figure 2 continued- Interaction of the distal tail segment of B-RafOUT with the active site of B-RafIN.
(D) Comparison of the structure of the B-Raf:14-3-3 complex with that of the CDK2:Cyclin A:p27Kip1 complex (PDB ID: 1JSU) (33), and the autoinhibited form of twitchin kinase (PDB ID: 1KOB) (34). In the B-Raf:14-3-3 complex, the distal tail segment of B-RafOUT (magenta) enters the ATP-binding site of B-RafIN (cyan). In the CDK2:Cyclin A:p27Kip1 complex, the p27Kip1 inhibitor (magenta) enters the ATP-binding site of CDK2 (cyan). In twitchin kinase, the C-terminal tail of the kinase (magenta) enters the ATP-binding site. Selected hydrophobic sidechains in the inhibitory segments are shown as spheres.
Figure 3 - Mutational analysis of B-Raf.
(A) Left, schematic diagram of the cryo-EM structure, indicating the B-Raf variants that were analyzed. Right, schematic diagram of the B-Raf-ΔNΔDTS:14-3-3 complex, which lacks the N-terminal region and the distal tail segment (dotted circle).
(B-G) Relative levels of phospho-ERK (pERK) for cells expressing B-Raf variants. Mean values for relative pERK levels and standard deviations were plotted from three flow cytometry experiments (the complete histograms for pERK levels in the experiments are shown in Fig. S9B). For each experiment, the pERK level for unstimulated wild-type EGFP-B-Raf transfected cells at 0 min was set to 1, and all other values were normalized to this. The statistical significance of each measurement is indicated by ns (p value>0.05), * (p value <=0.05), ** (p value<=0.01), *** (p value<= 0.001).
Figure 3 continued - Mutational analysis of B-Raf.
(H) SDS-PAGE gel analysis of B-Raf constructs purified from HEK293T cells. M – Precision Plus Protein Unstained Standards (Bio-Rad); 1 – B-Raf-WT; 2 – B-Raf-ΔDTS; 3 – B-Raf-ΔN; 4 – B-Raf-δNΔDTS.
(I) Western blot analysis of MEK1 phosphorylation by B-Raf constructs with the N-terminal regulatory region present and without the N-terminal regulatory region. Coomassie brilliant blue staining of the membrane shows the total amount of MEK1 protein loaded to each lane on the gel.
Figure 4 - Molecular dynamics simulations of the B-Raf:14-3-3 complex.
(A) Instantaneous structures from two representative simulations are shown. Left, initial structure. Middle, structure after 500 ns, for one of the simulations with the distal tail segment intact. Right, structure after 6 ns, for one of the simulations with the distal tail segment deleted. Orange dashed circles indicate a region of close contact between B-RafIN and 14-3-3 in the initial structure.
(B) Disruption of the B-Raf dimer interface in one of the simulations in which the distal tail segment of B-RafOUT was deleted. The interface between the kinases is shown for the initial structure (left) and the structure after 500 ns of simulation (right).
(C) Interactions between the C-terminal tails of 14-3-3 and the B-Raf kinase domains. Shown here is a superposition of the backbone structures of the 14-3-3 tails (yellow) for three simulations with the distal tail segment of B-RafOUT intact, sampled every nanosecond over 500 ns. The 14-3-3 tails cluster around the C-lobes of the two B-Raf kinase domains. This occurs due to electrostatic complementarity, with each instantaneous structure forming two to three ion pairs between each tail segment and the adjacent kinase domain (Fig. S15).
(Click on the small image to get a higher-resolution version.)
Supplementary Figure 1 - MEK1 phosphorylation by purified full-length B-Raf.
SDS-PAGE gel analysis of MEK1 phosphorylation with or without addition of purified full-length B-Raf. Kinase-dead MEK1 D190N mutant (residues 37-393) was analyzed with or without B-Raf. The reaction was initiated by the addition of ATP to the solution. Aliquots were taken out for SDS-PAGE analysis at indicated time points, and the kinase reaction was quenched by the addition of SDS-loading buffer supplemented with EDTA. The result was analyzed by western blotting using phospho-MEK antibody first (top panel) and the same membrane was stained by Coomassie Brilliant Blue (bottom).
Supplementary Figure 2 - Cryo-EM image processing.
(A) Representative electron micrograph.
(B) Representative 2D class averages obtained from reference-free 2D classification.
(C) Summary of classification and refinement procedures.
Supplementary Figure 2 continued - Cryo-EM image processing.
(D and E) Comparison of the cryo-EM maps for the B-Raf:14-3-3 complex, with or without 3D classification following signal subtraction of the 14-3-3 density. The results of 3D auto-refinement before (panel A) and after (panel B) 3D classification with 3D mask at the kinase domain are shown. The 14-3-3 density was subtracted from the particle images by Relion 3.0 before the 3D classification. The unsharpened map is shown here, and the cryo-EM map was colored the same way as Fig. 1B. The density corresponding to the distal tail segment of
B-RafOUT is highlighted by a circle.
(F) Fourier shell correlation (FSC) curves obtained from Relion 3 postprocess job. The resolution is estimated at FSC = 0.143.
(G) Fourier shell correlation (FSC) curves obtained from Phenix refinement job.
(H) The angular distribution of the final reconstruction obtained from Relion.
Supplementary Figure 3 - Cryo-EM reconstruction of the B-Raf:14-3-3 complex.
(A) The cryo-EM map of the B-Raf:14-3-3 complex colored the same way as Fig. 1B.
(B) The cryo-EM map of the B-Raf:14-3-3 complex colored by the local resolution estimated by Relion 3 (40) is shown in two orthogonal views at the top. The color scale corresponding to the local resolution is shown above the electron density maps. The cryo-EM model of the B-Raf:14-3-3 complex is shown below the corresponding views of the cryo-EM map.
Supplementary Figure 4 - Correspondence between the cryo-EM map and the crystal structures used to build the cryo-EM model.
(A)Coordinates for the B-Raf kinase dimer are taken from the crystal structure of the B-Raf:MEK complex (PDB ID: 4MNE) (22) and fit into the cryo-EM map using chimera (61). Helix G of B-RafOUT has weak density and is shown in gray.
(B) A crystal structure of a 14-3-3s and Raf C-terminal peptide complex (PDB ID: 4IEA) (47) is fit into the cryo-EM map corresponding to each 14-3-3 molecule in the cryo-EM map, respectively.
(C) The comparison of the angles between two subunits in the 14-3-3 dimers. Structural models of fifteen human 14-3-3z homodimers (gray) from 12 different crystal structures with unique space group (PDB IDs: 1IB1, 1QJA, 2C1N, 3RDH, 4HKC, 4N7G, 4ZDR, 5D2D, 5D3F, 4N7Y, 6EJL, 6F09) as well as 14-3-3s homodimer with the Raf C-terminal phosphopeptide (PDB ID: 4IEA, green) and the cryo-EM structure (red) were aligned using only one protomer (dashed line). The positions of the other protomer varies relative to the aligned protomers and the variation is highlighted by the arrow.
Supplementary Figure 5 - Interactions formed by the B-Raf C-terminal tail.
Interactions of the proximal tail segments of B-RafOUT (A) and B-RafIN (B) with 14-3-3. The 14-3-3 binding elements of each B-Raf molecule are also shown. Ins-1 and Ins-2 indicate (GSGSGS) residues insertion sites to generate the mutants for the cell-based assay. (A) The sidechain of Thr227 of 14-3-3 and the carbonyl group of Arg719, the last turn of helix αI in B-RafOUT, are located in close proximity.
(C) Disposition of the B-RafOUT distal tail segment with respect to the docking of the B-Raf tail on 14-
3-3. The distal tail segment is highlighted in green.
(D) Comparison of the positions of the B-RafOUT distal tail segment in the B-Raf:14-3-3 complex, the small molecule inhibitor, SB-590885, in the B-Raf dimer (PDB ID: 2FB8) (21), and the ATP molecule in the prototypical PKA structure (PDB ID: 1ATP) (52).
Supplementary Figure 6 - Cryo-EM density for the 14-3-3 binding element and the distal tail segment of B-RafOUT.
The structural model and the cryo-EM density corresponding to the C-terminal tail of B-RafOUT. Residues from Arg726 to Ala749 are shown, spanning the 14-3-3 binding element (orange) and the distal tail segment (magenta).
Supplementary Figure 7 - Comparison of B-RafIN and B-Raf Cdk/Src OFF-state.
The cryo-EM structure of the B-Raf:14-3-3 complex (cyan, magenta and gray) and the structure of B-Raf in the Cdk/Src OFFstate conformation (orange; PDB ID: 4WO5) (16) are superimposed on the kinase domains. A small molecule inhibitor that is bound to the kinase domain in the Cdk/Src OFF-state conformation
has been removed for clarity. The surfaces of the kinase domains are shown in the two closeup views. In the cryo-EM structure, note that the distal tail segment of B-RafIN (cyan) packs closely within the active site of the kinase domain of B-RafOUT (magenta). In the Cdk/Src OFF-state, closure of the N-lobe of the kinase domain against the C-lobe would lead to severe steric clashes with the distal tail segment.
Supplementary Figure 8 - Schematic diagram of the cell-based assay.
Ba/F3 cells were transfected with peGFPN1 vector, to make B-Raf protein fused to EGFP at the N-terminus. The cells were sorted by different EGFP expression levels using flow cytometry, and the distribution of the cells with various phospho-ERK levels is graphed.
Supplementary Figure 9 - Analysis of pERK levels upon expression of B-Raf.
(A-J) Histograms of Ba/F3 cells with different phospho-ERK levels. The Ba/F3 cells were transfected with N-terminally EGFPtagged wild-type B-Raf protein (A) or B-Raf variants (B-J) (blue lines) to compare to the endogenous signaling strength (GFP negative; gray lines). Each experiment was repeated more than three times and three representative results are shown here.
Supplementary Figure 10 - Sequences of the C-terminal tail segments of Raf.
Shown here is an alignment of the sequences of the C-terminal tail segments of B-Raf homologs from 14 metazoan species at the top, and human A-Raf and C- Raf at the bottom. αI is the last alpha-helix of the kinase domain. Species in the sequence alignments are Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Strongylocentrotus purpuratus, Lingula anatine, Lottia gigantea, Drosophila
melanogaster, Anopheles gambiae, Trichinella papuae, Trichuris suis, Nematostella vectensis, and Amphimedon queenslandica.
Supplementary Figure 11 - Analysis of the effect of mutations in the distal tail segment of B-Raf.
(A) Close-up view of active site of B-RafIN (cyan) with the distal tail segment of B-RafOUT (magenta).
(B) Sequence of the portion of the B-Raf distal tail segment that interacts with the active site of B-RafIN. Two Raf variants, denoted LYGG and GGSSGG, correspond to changes in the sequence as shown.
(C and D) Relative pERK levels for Ba/F3 cells expressing these B-Raf variants. Mean values for relative pERK levels and standard deviations were plotted from three flow cytometry experiments (the complete histograms for pERK levels in the experiments are shown in Fig. S9). Mean relative pERK levels and their standard deviations were plotted by the same way as Fig. 3. No statistical significance was observed in this experiment, as indicated by ns (p value>0.05).
Supplementary Figure 11 continued - The result of the repeated experiment shown in Fig. 3H and I.
(E) SDS-PAGE gel analysisof B-Raf constructs purified from HEK293T cells. M – Precision Plus Protein Unstained Standards (Bio-Rad); 1 – B-Raf-WT; 2 – B-Raf-ΔDTS; 3 – B-Raf-ΔN; 4 – B-Raf-ΔNΔDTS.
(F) Western blot analysis of MEK1 phosphorylation with the N-terminal regulatory region present and without the N-terminal regulatory region. Coomassie brilliant blue staining of the membrane shows the total amount of MEK1 protein loaded to each lane on the gel.
Supplementary Figure 12 - Stability of the B-Raf:14-3-3 complex structure in molecular dynamics simulations.
(A) The structural diagram shows the active site of B-RafIN (cyan), with the distal tail segment of B-RafOUT (magenta). The hydroxyl group of Tyr746 makes two hydrogen bonds with the hinge connecting N- and C-lobes of the kinase (dotted lines). The graphs show the time evolution of the distance between the oxygen of the tyrosine sidechain and the carbonyl of Gln530 (cyan) and an amide nitrogen of Cys532 (peach). In instantaneous structures sampled from the trajectories, at least one of the hydrogen bond is maintained. The darker traces are the time-averaged values of the distances calculated using a moving window of 4 ns.
(B) Stability of the asymmetric conformation when the B-RafOUT distal tail segment present or absent. Time series of the distance between the Cα atom of Arg462, in the N-lobe of B-RafIN and the Cα atom of Glu208 in 14-3-3. In the system with the distal tail segment present (left) the N-lobe of B-RafIN stays closely associated with 14-3-3, whereas in the system where the distal tail segment is absent (right), B-RafIN samples a range of conformations across the three simulations.
Supplementary Figure 13 - Stability of the B-Raf dimer interface in molecular dynamics simulations.
Distance between two B-Raf protomers are plotted during molecular dynamics simulations with or without B-RafOUT distal tail segment. The graphs show the time evolution of the distance between the Cα of His477 of B-RafIN and the Cα of Asp565 of B-RafOUT (cyan) and the distance between the Cα of His477 of B-RafOUT and the Cα of Asp565 of B-RafIN (peach). Red arrow indicates the breakage of the B-Raf dimer interface. The lighter shades are the actual distance and the darker curve represents the time-average of the distance, calculated with a period of 4 ns.
Supplementary Figure 14 - Sequences of the C-terminal tails of 14-3-3 proteins.
Alignment of the sequences of the C-terminal region of the seven human isoforms and the two insect cell 14-3-3 proteins, generated using Jalview (62). The residues corresponding to the last α-helix of 14-3-3, αI, are indicated. The residues are highlighted using the following color scheme: basic residues in blue, acidic residues in red, polar residues in green, hydrophobic residues in salmon, cysteines in yellow, tryptophans in dark yellow, and glycines in purple.
Supplementary Figure 15 - The interaction between the B-Raf kinase domain and the 14-3-3 C-terminal tail.
B-Raf kinase domain presents positively charged surfaces near the C-terminal tails of 14-3-3, which contain multiple acidic residues (see Fig. S14). The number of ion pairs between B-Raf and the 14-3-3 C-terminal tail was counted during the simulations and shown as histograms.
Supplementary Figure 16 - SDS-PAGE gel analysis of the B-Raf samples.
(A) Proteins produced during the production of B-Raf, analyzed by SDS-PAGE gel electrophoresis with Coomassie Brilliant Blue staining. showing the purification processes. M – Precision Plus Protein Unstained Standards (Bio- Rad); 1 - purified MBP-B-Raf-N fragment; 2 - MBP-B-Raf-N fragment after TEV protease treatment; 3 - purified B-Raf-C fragment; 4 - the product of intein reaction combining the B-Raf-N and B-Raf-C fragments; and 5 - purified full-length B-Raf.
(B) Western blot analysis of the purified full-length B-Raf protein, the product of intein chemistry, with phospho-C-Raf (Ser259) antibody, which also detects B-Raf Ser365 phosphorylation (39). The purified B-Raf protein was incubated with PKA and ATP for indicated time at 30°C, and subsequently run on an SDS-PAGE gel.
(C) Size exclusion chromatography profile of full-length B-Raf purification on Superose 6 column after intein reaction and TEV protease cleavage.
Supplementary Figure 17 - Sequence conservationin 14-3-3 proteins.
(A) Sequence alignment of human and insect 14-3-3e and z proteins. The conserved residues among all four genes are highlighted with red.
(B) Mapping conserved residues on the 14-3-3 structure. Crystal structure of 14-3-3s in complex with the Raf C-terminal peptide (PDB ID: 4IEA) (47) is shown in surface representation. The residues conserved among human and S. litura 14-3-3ε and ζ proteins are colored red and the others are gray. The position of the B-Raf dimer is indicated by dashed circle in the left panel.