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

Abstract

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins. 1-3 Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices. 4,5 They are activated by BH3-only proteins binding to the α2-α5 surface groove, 6-12 or for Bax, to the α1/α6 'rear pocket'. 13 Binding triggers dissociation of the latch domain (α6-α8) from the core domain (α2-α5), together with exposure of N-terminal epitopes and the BH3 domain. 6,7,14-16 The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 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.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the...

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

“…A fusion of the Bax TMD to the C terminus of the biotin ligase BirA (BirA/BaxTMD) biotinylated endogenous Bax and Bcl-x L in nonapoptotic HCT116 cells ( interactions. Our results are supported by Bak (and Bax) TMD interfaces mediating homooligomerization in the absence of BH3-dependent interactions (45). Mutant Bax proteins lacking either alpha 5 (Δ5) or alpha 6 (Δ6) helices, which are potentially relevant for membrane insertion and pore formation (19,26,28,46), retained the capacity for Bax association in mitochondrial membranes (Fig.…”

“…Lack of inhibition by the Bax TMD is particularly interesting, because the peptide concentration exceeded the native Bax protein concentrations. Therefore, symmetric Bax, and perhaps Bak oligomers, as recently suggested (27,45), could tolerate the association of multiple TMDs. Alternatively, other oligomeric Bax structures could be more prominent in OMM permeabilization.…”

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

“…Consistent with this conclusion, a recent study with reconstituted membranes suggested that, once in the membrane, Bax molecules form dimers, which further self-assemble into homo-oligomers (Subburaj et al 2015). Furthermore, an α9-α9 interface was recently detected in the homo-oligomers of Bax and Bak (Bleicken et al 2014;Gahl et al 2014;Iyer et al 2015). It can be speculated that, while the OMM lipids (e.g., cardiolipin, and sphingolipid products) are potential candidates to directly activate Bax/Bak, the protein components may play a modulatory role, as the OMM proteins Drp-1, OPA-1, Mfn1, etc., have been shown to play a role in MOMP (Scorrano et al 2002;Montessuit et al 2010;Renault et al 2015).…”

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

“…Similar experimental approaches were used to confirm the membrane targeting function of the C-terminus, but also suggest that the tail-anchor regions of Bcl-2 family proteins undergo a novel intramembrane dimerization (Andreu-Fernandez et al, 2017; Bleicken et al, 2014) where the C-terminal transmembrane domain, helix α9, of two Bax molecules interact either in parallel or anti-parallel angles within the membrane and away from the apoptotic pore (Bleicken et al, 2014; Iyer et al, 2015; Liao et al, 2016). C-terminal dimer interactions within the mitochondrial membrane were not required for release of small molecules, such as cytochrome c , but were needed to form larger pores, suggesting that these tail-tail interactions are required for pore expansion (Zhang et al, 2016).…”