A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

Robust autoactivation for apoptosis by BAK but not BAX highlights BAK as an important therapeutic target.

BAK and BAX, which drive commitment to apoptosis, are activated principally by certain BH3-only proteins that bind them and trigger major rearrangements. One crucial conformation change is exposure of their BH3 domain which allows BAK or BAX to form homodimers, and potentially to autoactivate other BAK and BAX molecules to ensure robust pore formation and cell death. Here, we test whether full-length BAK or mitochondrial BAX that are specifically activated by antibodies can then activate other BAK or BAX molecules. We found that antibody-activated BAK efficiently activated BAK as well as mitochondrial or cytosolic BAX, but antibody-activated BAX unexpectedly proved a poor activator. Notably, autoactivation by BAK involved transient interactions, as BAK and BAX molecules it activated could dissociate and homodimerize. The results suggest that BAK-driven autoactivation may play a substantial role in apoptosis, including recruitment of BAX to the mitochondria. Hence, directly targeting BAK rather than BAX may prove particularly effective in inhibiting unwanted apoptosis, or alternatively, inducing apoptosis in cancer cells.

BAX Activation: Mutations Near Its Proposed Non-canonical BH3 Binding Site Reveal Allosteric Changes Controlling Mitochondrial Association.

To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.

Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage.

Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.

Disordered clusters of Bak dimers rupture mitochondria during apoptosis.

During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore.

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane.

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.