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

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Science  04 Oct 2019:
Vol. 366, Issue 6461, pp. 109-115
DOI: 10.1126/science.aay0543

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The yin and yang of Raf inhibition

Many human melanomas contain an overactive form of Raf kinase (B-Raf). Inhibitors are effective against the mutant B-Raf, but, paradoxically, they activate wild-type B-Raf, limiting their therapeutic potential. Kondo et al. determined the structure of a phosphorylated B-Raf dimer in complex with the scaffold protein 14-3-3 by cryo–electron microscopy. Although both kinases are in the active conformation, one is blocked by the C-terminal tail of the other. This configuration inhibits one active site but also stabilizes the dimer in the active conformation. Understanding this mechanism provides a framework for development of inhibitors that do not activate wild-type Raf.

Science, this issue p. 109


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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