Membrane insertion of the BAX core, but not latch domain, drives apoptotic pore formation

Abstract

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.

Topology of active, membrane-embedded Bax in the context of a toroidal pore

“…Most positions were highly water exposed, with one (A107C) being inaccessible. The accessibility results of the other groups mainly agree with the high water accessibility, with the exception of Flores-Romero et al [76]. The data on helix 5 are less consistent, and especially mutations on the conserved amino acids N106, V111, F116, and L122 are problematic ( Fig.…”

“…In contrast, Zhang et al [34] studied five positions and found them all to be < 50% water exposed, whereas EPR studies suggested four positions involved in protein-protein contacts [32,50,67]. Finally, Flores-Romero et al [76] detected a low water and a low lipid accessibility for two positions in helix 2, also in line with a protein contact area. An example of data inconsistencies is the following: E69C (in Bax, D84C in Bak) was found to be 100% water accessible in Uren et al [39], 10% accessible in Zhang et al [34], and a third study described the D84G mutation to have a loss-of-function phenotype [65] (more examples in the Supplementary Tables).…”

“…Amino-acid conservation highlighted by green (identical amino acid) or orange (conserved amino acid) stars on the upper left corner of the box. The water accessibility of each amino acid based on the available data ( [49,37,39,47,34,50,68,52,76] and our data) is indicated by the gray letters in the upper right corner of the box (classified as high (H), high to moderate (H/M), moderate (M), moderate to low (M/L) and low (L) water accessibility). The symbol "?"…”

BFL1 modulates apoptosis at the membrane level through a bifunctional and multimodal mechanism showing key differences with BCLXL

“…In further SEC experiments, the canonical BFL1ΔC R88D mutant behaved as the native protein further supporting that BFL1ΔC forms homocomplexes via noncanonical interactions, unlike the case of BAX (Fig. S4b) [2, 29].

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“…We used a recently validated set of NBD-labeled BAX monocysteine variants encompassing the canonical BAX BH3 motif (Fig. 1f, red) [29], and the noncanonical BAX surface that binds the BCL2 BH4 motif (Fig. 1f, cyan) [8].…”