To test whether priming was mediated by inhibitor’s conformational effects on RAF kinase domain
Experiment 1 and result
Both purified recombinant protein and non-BRAF (V600E) cell lines show the formation of a stable complex between CRAF and BRAF kinase domain.
Using immunoprecipitation and western blot.( showing how much CRAF binds to BRAF.)
Using immunoprecipitation and western blot.( showing how much CRAF binds to BRAF.)
Treatment of inhibitors:
Non-hydrolysable analogue of ATP and PLX4720: destablise the complex
AZ-628 and GDC-0879: stablise the complex
Conclusion: inhibitors’ effects on RAF kinase domain mediate RAF dimerization.
Crystal structure of CRAF kinase domain complexed to a close analogue of GDC-0879
Description: a model of CRAF homodimer with inhibitor (shown in magenta). In the part of cartoon mode, non-covalent bonds are shown as yellow dot line. Monomer in surface mode showed the charge on the surface. The “black hole” between monomers illustrates a side-to-side interaction.
Binding site:
Description: A crucial residue on alpha C-helix shown in magenta interacts with the RAF inhibitor through non-covalent bond in binding site. This is a potential reason why inhibitor-binding (PLX4720) gives rise to a shift on alpha C helix, thus affect the dimer interaction and inhibitor priming.
Comparision of BRAF and CRAF homodimers:
1. An extremely well conserved interface
2. Differ only in the most N-terminal residue of CRAF within negative charged regulatory domain
3. Both are homodimers and interact through side-to-side manner.
4. Non-conserved residues between BRAF and CRAF are shown in violet. Conserved residues in the surface depiction are shown in green. Residues in the dimer interface are coloured in lime green.
To test whether CRAF kinase activity will be influenced by the modulation of kinase domain dimerization by RAF inhibitors
Generate CRAF and BRAF dimer interface mutants:
1) Constitutive dimerization: CRAF (E478K) and BRAF (E586K)
2) Defective dimerization: CRAF (R401H) and BRAF (R509H)
(Both kinds of the dimers are co-transfected into HCT116 KRAS-MT cell)
From 1)-- a basal increase of exogenous CRAF kinase activity compared to WT control.
From 2)-- Decreased level of basal CRAF activity
-- Impairment of CRAF activation by RAF inhibitor
Conclusion:
Kinase domain dimerization by RAF inhibitor underlies CRAF activity and downstream MEK signalling.
