Structural Model of Active Bax at the Membrane

Bleicken, Stephanie; Jeschke, Gunnar; Stegmueller, Carolin; Salvador‐Gallego, Raquel; García‐Sáez, Ana J.; Bordignon, Enrica

doi:10.1016/j.molcel.2014.09.022

Cited by 198 publications

(348 citation statements)

References 42 publications

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“…Our data show that during apoptosis, the N-segment and a1 of Bak dissociate entirely from the rest of the protein to expose the BH4 domain and other residues (Fig. 9b), consistent with recent reports that Bak a1 and a6 became 450 Å apart after tBid treatment in LUV 26 and the distance between Bax a1 and a2 varies widely after activation 21 . Disulfide tethering of Bak showed that a1 dissociation from both the core (a2-a5) and the latch (a6-a8) was required for pore formation.…”

Section: Discussionsupporting

confidence: 92%

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Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

et al. 2015

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During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the a1 helix are exposed indicating complete dissociation of a1 from a2 in the core and from a6-a8 in the latch. Moreover, disulfide tethering of a1 to a2 or a6 blocks cytochrome c release, suggesting that a1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when a1 is tethered shows that Bid triggers a2 movement, followed by a1 dissociation. However, a2 reaches its final position only after a1 dissociates from the latch. Thus, a1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (a2-a5) and the latch (a6-a8).

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Section: Discussionsupporting

confidence: 92%

“…Another key conformation change identified in Bak and Bax is separation of the latch (a6-a8) from the core (a2-a5) 19,20 . After these large changes in conformation, the proteins form BH3:groove dimers and make significant contact with the outer membrane surface 9,16,19,21,22 .…”

mentioning

confidence: 99%

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

et al. 2015

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Section: Discussionmentioning

confidence: 94%

“…29,67 Furthermore, the α6-α9 region in oligomerized Bax was found to be extended 29 and flexible. 67 Notably, Bleicken et al 67 suggested the α9:α9 interaction 'within' dimers may be anti-parallel based on a model in which the α2-α5 core dimers position on the rim of the pore rather than facing the cytosol. Although our linkage studies and the FRET studies of Gahl et al 29 show parallel interaction of the α9 helices, we may be detecting interactions 'between' dimers (as depicted in Figure 7d) and not the interactions that might occur 'within' dimers.…”

Section: Discussionmentioning

confidence: 94%

“…An α9:α9 interface in Bak (and Bax) oligomers was demonstrated by linkage of the α9 helices after pore formation, consistent with very recent evidence of an α9:α9 interface in Bax oligomers, Figure 3a, or for cytochrome c release based on Förster resonance energy transfer (FRET) 29 and crosslinking. 67 A distinct cysteine linkage pattern along α9 was not due to a dimerization domain, but may be caused by a preferred packing surface between the helices and/or by limited rotation of α9 within oligomers. Linkage at Bak α9:α9 could link BH3:groove dimers to the higher-order oligomers that are associated with pore formation.…”

Section: Discussionmentioning

confidence: 95%

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

et al. 2015

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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...

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EPR Spectroscopy of Nitroxide Spin Probes

Bordignon

2017

eMagRes

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Structural Model of Active Bax at the Membrane

Cited by 198 publications

References 42 publications

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

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

EPR Spectroscopy of Nitroxide Spin Probes

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