Ha, B. H.; Tkacik, E.; Gazgalis, D.; Kang, H.; Jang, D. M.; Chakraborty, S.; Jeon, H.; Eck, M. J.

Abstract Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero- dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.