Phosphorylation of RAF kinase dimers drives conformational changes that facilitate transactivation

Abstract

RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N-terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC-helix and charged residues upstream of the NtA motif. Additionally, we show that the R-spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure-based mechanism for the auto-transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization-related drug resistance. Back to back: Biochemical kinase assays and structural evidence from MD simulations reveal a key electrostatic role for the phosphorylated NtA motif in RAF kinase transactivation. Interprotomer salt bridges and a conserved tryptophan residue located at the N-terminal end of the kinase domain play crucial roles by connecting the R-spines of the two protomers, and phosphorylation leads to important structural changes in the highly conserved HRD motif.