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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📊 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, re...
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 s...
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-u0394Nu0394DTS:14-3-3 complex, which lacks the N-terminal ...
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. Rig...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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