A Redox Switch Regulates the Structure and Function of Anti-Apoptotic BFL-1

BCL-2 family proteins control cell fate by regulating the mitochondrial apoptotic pathway. Anti-apoptotic members suppress cell death by capturing pro-apoptotic α-helices in a surface groove, a mechanism hijacked by cancer cells to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the canonical groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the interface between the groove (C55) and C-terminal α9 helix (C175) operates as a redox switch to control the accessibility and functionality of the anti-apoptotic pocket. Hydrogen deuterium exchange mass spectrometry was used to characterize a construct of BFL-1 deleting the C-terminus and thus C175 (BFL-1ΔC C55), as well as full length BFL-1 (BFL-1 C55/C175) in both oxidation states. Reducing the C55-C175 disulfide bond triggers a cascade of structure-function changes that includes release of α9 for membrane translocation, exposure of the groove for α-helical interaction, and resultant blockade of mitochondrial membrane permeabilization by pro-apoptotic BAX. Thus, we identify a unique mechanism of conformational control over anti-apoptotic activity by a redox switch in BFL-1.