Dimeric BRAF kinase domains bound to dimeric scaffold protein 14-3-3 had active-site asymmetry.

  • Major Finding: Dimeric BRAF kinase domains bound to dimeric scaffold protein 14-3-3 had active-site asymmetry.

  • Concept: The C terminus of one kinase occludes the other's active site, so only one is capable of catalysis.

  • Impact: The structure improves our understanding of paradoxical activation of RAFs by some BRAF inhibitors.

Although inhibitors of RAF kinases have been successfully developed as cancer drugs, many BRAF inhibitors have unexpectedly been found to activate RAF signaling. This effect may be due to homo- and heterodimerization of RAF kinases: At sub-saturating concentrations, an inhibitor-bound RAF kinase may dimerize with an inhibitor-free partner, an interaction that stabilizes the active (“on”) state of the latter. Expanding our understanding of this phenomenon, Kondo, Ognjenović, and colleagues used cryo-electron microscopy to determine the structure of an active, dimeric BRAF kinase domain in complex with dimeric 14-3-3, a scaffold protein that facilitates RAF kinase domain dimerization. The structure, with an overall resolution of 3.9 Å, revealed asymmetry in the kinase dimer, despite the fact that both kinase domains were in the active (“on”) state. Insertion of the C-terminal tail of one kinase domain physically occluded the active site of the other kinase domain, meaning that only one of the two kinases would be capable of performing catalysis. Additionally, the symmetry axes of the kinase dimer and the 14-3-3 dimer, both of which exhibit twofold symmetry on their own, were not aligned in the full complex; the active site of one kinase domain was closer to 14-3-3 than that of the other kinase domain. Experiments in an IL3-dependent mouse hematopoietic cell line often used in drug discovery for kinase inhibitors showed that the exact positioning of the BRAF dimer relative to the 14-3-3 dimer was critical for phospho-ERK signaling, which is a downstream consequence of RAF signaling. Further, biochemical assays revealed that the presence of the BRAF kinase domain's distal tail segment was important for MEK1 phosphorylation. Molecular dynamics simulations provided evidence that the interactions between one kinase domain and the distal tail segment of the other kinase domain are responsible for the asymmetry in the positioning of the kinase domains and 14-3-3 proteins. Together, these findings strengthen our understanding of BRAF activation, providing a framework for the development of new drugs targeting this pathway.

Kondo Y, Ognjenović J, Banerjee S, Karandur D, Merk A, Kulhanek K, et al. Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases. Science 2019 Sep 19 [Epub ahead of print].

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