.2019 Oct 4;366(6461):109-115.

doi: 10.1126/science.aay0543. Epub 2019 Sep 19.

Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Yasushi Kondo^#^{1 2}, Jana Ognjenović^#³, Saikat Banerjee⁴, Deepti Karandur^{1 2 5}, Alan Merk³, Kayla Kulhanek⁴, Kathryn Wong^{1 2}, Jeroen P Roose⁴, Sriram Subramaniam⁶, John Kuriyan^{7 2 5 8 9}

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20814, USA.
⁴ Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. kuriyan@berkeley.edu sriram.subramaniam@ubc.ca.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. kuriyan@berkeley.edu sriram.subramaniam@ubc.ca.
⁸ Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁹ Divisions of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

^# Contributed equally.

PMID:31604311
PMCID: PMC7007921
DOI: 10.1126/science.aay0543

Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Yasushi Kondo et al. Science.2019.

.2019 Oct 4;366(6461):109-115.

doi: 10.1126/science.aay0543. Epub 2019 Sep 19.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20814, USA.
⁴ Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. kuriyan@berkeley.edu sriram.subramaniam@ubc.ca.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. kuriyan@berkeley.edu sriram.subramaniam@ubc.ca.
⁸ Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁹ Divisions of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

^# Contributed equally.

PMID:31604311
PMCID: PMC7007921
DOI: 10.1126/science.aay0543

Abstract

Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical "active" conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.

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Conflict of interest statement

Competing interests: Jeroen Roose is a co-founder and scientific advisor of Seal Biosciences, Inc. and on the scientific advisory committee for the Mark Foundation for Cancer Research. John Kuriyan is a co-founder of Nurix Therapeutics, Inc., and on the Scientific Advisory Boards of Carmot, Inc., and Revolution Medicine, Inc.

Figures

**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-Raf^IN (cyan) is positioned closer to 14-3-3 than is B-Raf^OUT (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) (Fig S4). Helix αG of B-Raf^OUT 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-Raf^OUT with the active site of B-Raf^IN.**
(A) Orthogonal views of the cryo-EM structure of the B-Raf:14-3-3 complex. On the left, the C-tail of B-Raf^OUT (magenta) is seen bound to 14-3-3 (gray) and the distal tail segment enters the active site of B-Raf^IN (cyan). (B) View of the ATP binding site of B-Raf^IN (cyan), with cryo-EM density shown in gray. Residues in the distal tail segment of B-Raf^OUT (magenta) are identified by asterisks. (C) The hydrophobic sidechains of the C-spine of B-Raf^IN are shown as yellow spheres, with two sidechains of the B-Raf^OUT distal tail segment (magenta) completing the C-spine. (D) Comparison of the structure of the B-Raf:14-3-3 complex with that of the CDK2:Cyclin A:p27^Kip1 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-Raf^OUT (magenta) enters the ATP-binding site of B-Raf^IN (cyan). In the CDK2:Cyclin A:p27^Kip1 complex, the p27^Kip1 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). (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-Raf^IN 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-Raf^OUT 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-Raf^OUT 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).

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References

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Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Affiliations

Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

Research Materials

Miscellaneous