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.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x_L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization.