An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane.
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ABSTRACT:
SUBMITTER: Lv F
PROVIDER: S-EPMC8280806 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.
| S-SCDT-EMBOJ-2020-106438 | biostudies-other
Project description:Despite intensive research effort, how the paradigmatic proapoptotic protein BAX forms lethal apoptotic pores at the mitochondrial outer membrane (MOM) remains incompletely understood. Here, we used biophysical tools and minimalist model systems to identify the specific regions in BAX driving apoptotic pore formation, and to gain more insight into underlying mechanisms. Fluorescence mapping revealed that fully active BAX adopts a BH3-in-groove dimeric conformation in MOM-like membranes, with BAX α4-α5 helices belonging to its core domain inserting deeper into the membrane lipid bilayer than BAX α6-α8 helices belonging to its latch domain. In our reconstituted systems, antiapoptotic BCLXL formed canonical heterodimeric BH3-in-groove complexes with BAX, and blocked membrane insertion of BAX core α4-α5 helices, but not BAX latch α6-α8 helices. Moreover, poly(ethylene glycol) (PEG) conjugation (PEGylation) at multiple individual sites along the BAX core, but not latch domain, potently inhibited BAX pore-forming activity. Additional combined computational and experimental evidence revealed that the BAX core α5 helix displays a bilayer-destabilizing membrane interaction mode that is absent in BAX latch α6-α8 helices. Based on this collective set of evidence, we propose that membrane insertion of the BAX core, but not latch domain, is critical for BAX apoptotic pore formation.
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Project description:Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model - using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.
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Project description:Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.
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Project description: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.
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Project description:The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.
| S-EPMC1222950 | biostudies-other
Project description:Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVLTASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an ?/?-type secondary structure within membranes. Both ?-strands and ?-helices are significantly (by 30-60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8-9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an "?/?-ring" structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi.
| S-EPMC4537061 | biostudies-literature
Project description:How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement.
| S-EPMC4891688 | biostudies-literature
Project description:Fatty acids are known promoters of apoptosis. In the present study, the direct role of fatty acids with regard to their ability to cause membrane permeabilization by Bax was explored. Addition of fatty acids to liposomes in the presence of cations greatly enhanced the permeabilizing activity of Bax, a pro-apoptotic Bcl-2 protein. This provides a putative mechanism for the role of fatty acids in apoptosis. It is not a result of detergent-like properties of fatty acids, since a different micelle-forming amphiphile, dilysocardiolipin, was strongly inhibitory. We also demonstrate that there is a synergistic effect on Bax-induced permeabilization between Ca(2+) and Mg(2+), both on the binding of Bax to liposomes as well as on the induction of the leakage of liposomal contents. Micromolar concentrations of Ca(2+) added externally or submicromolar concentrations of free Ca(2+) present in the medium were sufficient to promote Bax-induced permeabilization synergistically with externally added Mg(2+). These results indicate that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically. The synergistic effects of Ca(2+) and Mg(2+) were observed with liposomes with different lipid compositions. Thus the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.
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Project description:BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.
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