Cryo-EM and biochemical characterization of a BRAF/CRAF heterodimer shows that the NtA motif negative charge is dispensable for RAF activation.

Cryo-EM structures and biochemical assays of 14-3-3–bound BRAF/CRAF heterodimer complexes show NtA acidic charge is dispensable for RAF activation, with RARA mutants forming active dimers.

• Why it matters: RAF heterodimers are important for physiologic and oncogenic MAP kinase signaling, yet their structural and biochemical basis—especially how NtA charge affects activation—remains poorly understood.
• What they did: The authors prepared 14-3-3–bound BRAF/CRAF heterodimer complexes, measured kinetic parameters and RAF inhibitor sensitivities, and solved cryo-EM structures; they then used mutagenesis to swap the acidic NtA sequence to RARA.
• The result: The heterodimer’s kinetics and inhibitor sensitivities are intermediate between, or closely matching, BRAF and CRAF homodimers, and the NtA-to-RARA change yields active dimers, implying NtA charge affects local backbone dynamics and inactive-state stability rather than stereospecific interface recognition.