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Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation

Nature Structural & Molecular Biology volume 22, pages 37–43 (2015)Cite this article

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Abstract

Reported RAF kinase domain structures adopt a side-to-side dimer configuration reflective of an 'on' state that underpins an allosteric mechanism of regulation. Atomic details of the monomer 'off' state have been elusive. Reinspection of the BRAF kinase domain structures revealed that sulfonamide inhibitors induce features of an off state, primarily a laterally displaced helix αC stabilized by the activation segment helix 1 (AS-H1). These features correlated with the ability of sulfonamides to disrupt human BRAF homodimers in cells, in vitro and in crystals yielding a structure of BRAF in a monomer state. The crystal structure revealed exaggerated, nonproductive positions of helix αC and AS-H1, the latter of which is the target of potent BRAF oncogenic mutations. Together, this work provides formal proof of an allosteric link between the RAF dimer interface, the activation segment and the catalytic infrastructure.

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Figure 1: Inhibitors that induce an off state–like conformation of the BRAF kinase domain are dimer breakers in solution.
Figure 2: Crystal structure of a BRAF monomer yields insights in the allosteric control by dimerization by the AS.
Figure 3: Dimerization and activity of oncogenic BRAF mutants V600E and V600K and their dependence on Lys507.

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Acknowledgements

We thank M. Gao from Bristol-Myers Squibb Molecular Structure & Design group for making BRAF expression constructs. N.T. is supported by an Ontario Graduate Scholarship and a Canadian Institutes for Health Research (CIHR) Canada Graduate Scholarship. H.L. is supported by a CIHR Banting Postdoctoral Fellowship. F.S. is supported as a Canada Research Chair. This work was supported by a grant from the Ministère de l'Économie, de l'Innovation et de l'Exportation du Québec to A.M. and an Impact Grant from the Canadian Cancer Society Research Institute to M.T. (702319) as well as by operating funds from the CIHR to M.T. (MOP119443) and F.S. (MOP36399).

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Author notes

  1. Neroshan Thevakumaran and Hugo Lavoie: These authors contributed equally to this work.

Authors and Affiliations

  1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Neroshan Thevakumaran & Frank Sicheri

  2. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Neroshan Thevakumaran & Frank Sicheri

  3. Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada

    Hugo Lavoie, Anne Marinier & Marc Therrien

  4. Molecular Discovery Technologies Department, Molecular Structure & Design Group, Bristol-Myers Squibb Research, Princeton, New Jersey, USA

    David A Critton & Andrew Tebben

  5. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Frank Sicheri

  6. Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada

    Marc Therrien

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Contributions

N.T., H.L., F.S. and M.T. designed the experiments and wrote the manuscript. N.T. performed AUC experiments and in vitro kinase assays. H.L. performed BRET and co-IP experiments. D.A.C. conducted protein production, crystallization and X-ray data collection. Structural analysis of the BRAF monomer was performed by D.A.C., A.T., N.T. and H.L. A.M. provided expert advice for the interpretation of compound binding mode.

Corresponding authors

Correspondence to Frank Sicheri or Marc Therrien.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sulfonamide inhibitors occupy the RAF-selective pocket and retain dimer-breaker ability at nonsaturating concentrations in vitro.

(a) Structure.sdf files of sulfonamide compounds were obtained from the PDB and displayed using ChemBioDraw 12.0. (b) Comparison of the two protomers in crystal structures of BRAF bound to PLX4720 (PDB ID 3C4C15) or PLX4032 (PDB ID 3OG716). Protomers in a helix “αC-out”, off-like state conformation (blue) have the “RAF-selective” pocket occupied. For the protomer displaying a helix “αC-in”, on state conformation (orange), the “RAF-selective” pocket is not occupied due to an alternate binding mode of the inhibitor arising from the indicated rotation of the terminal sulfonamide motif. (c) Sedimentation velocity AUC analysis of a solution of the BRAF kinase domain (25 μM of protein) in a titration series with saturating (40 μM) and non-saturating (2.5 μM and 10 μM) of the sulfonamide inhibitors PLX4720, PLX4032 or B0R.

Supplementary Figure 2 The monomer off-state structure of BRAF closely resembles the forced dimer sulfonamide co-structures.

(a) Hydrophobic spines representation of BRAF in the dimer on state, monomer off state and forced dimer intermediate-state (Top). Hydrophobic spines representation of CDK2 in on and off states (Bottom). (b) Binding mode of PLX4720 in the BRAF monomer structure (PDB ID 4WO5; this study) and forced dimer structure (PDB ID 3C4C15). (c) Dimer interface residues of the 24 on state dimers (orange) and 10 off-like state forced dimers (green).

Supplementary Figure 3 Crystal contacts in the BRAF monomeric structure in complex with PLX4720.

Protomer 1 and 2 and their symmetry related mates are shown in light and dark blue, respectively. The side-to-side dimer configuration of on state structure is shown in orange and yellow for reference.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 5534 kb)

Supplementary Data Set 1

Uncropped western blots and autoradiograms presented in the main figures (PDF 442 kb)

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Thevakumaran, N., Lavoie, H., Critton, D. et al. Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation. Nat Struct Mol Biol 22, 37–43 (2015). https://doi.org/10.1038/nsmb.2924

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