Regulation of Apoptosis “Beyond the Point of No Return”: Inhibition of Oligomeric BAX by an Antiapoptotic Dimer

Abstract

The protein interactions among pro- and anti-apoptotic members of the BCL-2 family regulates the critical control point of apoptosis induction, mitochondrial outer membrane permeabilization (MOMP). Proapoptotic BAX lies dormant in the cytosol as a latent monomer until triggered by cellular stress to transform into an oligomeric species, translocate to the mitochondria, and induce MOMP. Anti-apoptotic BCL-2 proteins inhibit BAX activation by trapping a “killer” motif, the BH3 a-helix, before it can become embedded at the interface of propagating BAX oligomers. We previously reported the production and characterization of a functional, homogeneous BAX oligomer (BAXO) species, formed upon transient detergent exposure. Here we find that BCL-w, an anti-apoptotic protein that likewise partitions between cytosol and mitochondria, is transformed into stable, elongated dimers (BCL-wD) upon analogous detergent treatment. Whereas incubation of full-length, monomeric BCL-w with BAXO has no BAX-inhibitory effect, dimeric BCL-w directly engages and dissociates BAXO, blocking BAX-mediated mitochondrial poration. Hydrogen deuterium exchange mass spectrometry studies revealed a series of discrete conformational change that occurs upon BCL-w dimerization and reciprocal structural impacts upon BCL-wD and BAXO engagement. Chemical crosslinking and mass spectrometry revealed intra- and intermolecular crosslinks that further defined structural proximities of the protein states. BCL-w a1 emerged as an especially dynamic region involved in both the dynamic transformation of BCL-w and its regulation of BAXO. A stapled peptide mimetic of BCL-w a1 recapitulated BAXO-inhibitory activity, highlighting its mechanistic role in BAX regulation. The a5-a6 region of BCL-w, in addition to potentially contributing to the elongated dimeric interface, was found to have biophysical features that contrasted with the hydrophobic and positively-charged properties of BAX a5-a6, which contributes to membrane disruption. Indeed, preliminary SAXS analyses demonstrated that the negative Gaussian curvature induced by BAXO and that causes membrane permeabilization can be counterbalanced by positive Gaussian curvature imposed by BCL-wD. Taken together, the findings that emerged from our production and characterization of BCL-wD elucidated a novel mode of regulation beyond what was previously considered the “point of no return” for BAX-mediated apoptosis induction.