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

Abstract

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.cell death | mitochondrial permeabilization | protein-membrane topology | membrane pores | cysteine-scanning mutagenesis C ommitment 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...

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

“…To identify α9 residues buried in the hydrophobic interior of the MOM, cysteine variants were labeled with the membrane-impermeable sulfhydryl reagent 4-acetamido-4ʹ-((iodoacetyl)amino)stilbene-2,2ʹ-disulfonic acid (IASD). 20,23,49 The two negative charges on IASD prevent its entry into hydrophobic regions, including membranes, protein interior and protein interfaces. 23 IASD efficiently labeled cysteine residues on the cytoplasmic side of the MOM (Figures 2a, e and g; G186C).…”

“…20,23,49 The two negative charges on IASD prevent its entry into hydrophobic regions, including membranes, protein interior and protein interfaces. 23 IASD efficiently labeled cysteine residues on the cytoplasmic side of the MOM (Figures 2a, e and g; G186C). IASD also strongly labeled cysteine placed at the far C terminus (Figure 2a, BakGGCK), probably by passing through channels 20,50 such as VDAC, which allows passage of metabolites up to 4000 Da.…”

“…In addition, we examined whether the α9 helix (or the α9:α9 interface) might be necessary for the step of pore formation, as α9 appears to be the only region that traverses the MOM before and after pore formation, [21][22][23] and α9 peptides have been proposed to be membranolytic with antitumor activity. 43,58 Thus, understanding α9 membrane topology may reveal novel insight for cancer therapy.…”

“…After Bak was activated, the peripheral α9 33 Insertion may be driven by activators binding the hydrophobic groove and releasing α9, but may be promoted by the membrane disturbance caused by collapse of activated Bak onto the MOM. 23 Somewhat surprisingly, Bak targeting and insertion could be severely affected by swapping the whole C terminus with that from other MOM tail-anchored proteins.…”

“…19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.…”