Abstract
B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.
Highlights
Much of the current thoughts on the core molecular mechanism by which B-cell lymphoma 2 (Bcl-2) family proteins
It is generally agreed that BH3-only proteins promote Bcl-2-associated X protein (Bax)-mediated membrane permeabilization and their synergistic effect suggests a role in direct Bax activation, the underlying mechanism is under intense study, and is often the subject of considerable debate.[2,4]
Minimal reconstituted systems that included lipid membrane, activator protein or BH3 interacting domain death agonist (Bid) BH3 peptides, as well as pro-survival protein (Bcl-xL) served as powerful systems to study Bax activation and membrane permeabilization.[6,8,9,13,15,16,17]. These studies allowed unraveling some of the critical steps involved in the mechanism of Bax-mediated mitochondrial outer membrane permeabilization (MOMP) and provided the first observations for the existence of Bax-induced pores
Summary
Much of the current thoughts on the core molecular mechanism by which Bcl-2 family proteins, . The exact temporal sequence of events remains elusive As part of this process, Bax oligomerization is thought to occur sometime after membrane association, leading to large, irregular mixed protein-lipidic pores such as those observed by electron cryo-microscopy (cryo-EM) of large unilamellar vesicles.[13] the detailed spatiotemporal, molecular mechanism of Bax-mediated pore formation remains to be further defined and the explicit structure of membraneembedded Bax and how it gives rise to a functional pore remains an active area of investigation. Here we provide direct evidence that Bax monomers, once activated by Bid BH3 peptide and incorporated in a lipid bilayer, induce the formation of pores and permeabilize the membrane bilayer
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