VDAC2 directly forms a stable membrane-associated complex with BAX.

A. Schematic of the experimental strategy. Full-length BAX was synthesized by cell-free expression in the presence of preformed His-tagged VDAC2-containing nanodiscs and purified by Ni-NTA affinity chromatography through the VDAC2 His tag. B. Western blot analysis of BAX following cell-free synthesis and affinity purification with 6His- VDAC2-containing nanodiscs or His-tagged empty nanodiscs. BAX co-elutes with VDAC2-containing nanodiscs but not with empty nanodiscs. C. Co-immunoprecipitation of WT BAX and VDAC2. D. WT BAX and VDAC1, following cell- free synthesis in the presence of VDAC2- or VDAC1-containing nanodiscs. Intact nanodiscs were immunoprecipitated using either a BAX-specific antibody (2D2) or an anti-His antibody recognizing VDAC2 and captured on Protein G Sepharose. After binding, the nanodiscs were solubilized with 0.5% NP-40 to disrupt the lipid bilayer while preserving direct protein–protein interactions. Resin without antibody served as a negative control. E. Sodium carbonate extraction (pH 10) of the purified VDAC2–BAX complex. Following cell-free synthesis in the presence of VDAC2-containing nanodiscs, complexes were immobilized on Ni-NTA resin through the VDAC2 His tag. After washing away unbound BAX, a sodium carbonate wash (pH 10) was used to detach peripherally associated BAX. The remaining membrane-associated complex was subsequently eluted with imidazole (EL). A fraction of BAX was released by sodium carbonate, whereas the majority remained associated with VDAC2-containing nanodiscs. F. SDS–PAGE analysis of the Ni-NTA-purified VDAC2–BAX complex showing VDAC2, BAX and the membrane scaffold protein MSP1D1. G. Size-exclusion chromatography profiles of VDAC2-containing nanodiscs (blue), soluble BAX (green) and the purified VDAC2–BAX complex (magenta). H. Western blot analysis of fractions collected from the size-exclusion chromatography of the VDAC2– BAX complex. I. Representative cryo-EM 2D class averages obtained from the final particle sets corresponding to SEC peak 2 (left) and SEC peak 1 (right). J. Ab initio cryo-EM reconstruction of a VDAC2 dimer reconstituted in an MSP1D1 nanodisc, obtained from particles corresponding to SEC peak 2. K. Ab initio cryo-EM reconstruction of a VDAC2 dimer reconstituted in an MSP1D1 nanodisc in complex with BAX, obtained from particles corresponding to SEC peak 1. VDAC2 is shown in blue, BAX in yellow, and the nanodisc density in grey.

Structural modeling and crosslinking identify α9-mediated engagement of BAX by VDAC2.

A. Structure of soluble inactive BAX (PDB: 1F16) highlighting the trigger site (orange), canonical groove (yellow), BH3 domain (red), and C-terminal α9 helix (blue). The AlphaFold3 prediction, shown in transparent grey, closely reproduces the experimental structure (RMSD = 2.46 Å +/- 0.26 Å; SFig. S1A). B. Representative AlphaFold3 model of the VDAC2–BAX complex colored according to pLDDT confidence values (scale bar on the right). 15 models from 3 independent predictions converged on the same α9-mediated interaction interface (SFig. S1B). C. BAX western-Blot after Ni-NTA affinity purification of VDAC2 nanodiscs following cell-free expression of BAX-Äá9 shows that BAX-Äá9 does not associate with VDAC2. D. CuPhe-mediated cysteine cross-linking of VDAC2 and BAX single-cysteine mutants validates the predicted α9-mediated interface. Right insets show close-up views of the predicted interaction surface. VDAC2 is displayed as a surface representation colored according to residue properties: acidic (red), basic (blue), uncharged polar (green), aliphatic (white), and aromatic (light pink). BAX helix α9 is shown in cartoon representation, with selected side chains displayed as sticks to illustrate their complementarity with the VDAC2 surface. Helix α9 contains a conserved GXXXA motif; the Cα and Cβ atoms of G179 and A183 are shown as grey van der Waals spheres. Residues mutated to cysteines for cross-linking validation (VDAC2 K121C/BAX T169C and VDAC2 Y78C/BAX I187C) are shown as black sticks. Left: Experimental validation of the α9 orientation by cysteine cross-linking. Western blots of complexes containing, from left to right, VDAC2 K121C/BAX T169C, VDAC2 Y78C/BAX I187C, VDAC2 ΔCys/BAX T169C, and VDAC2 ΔCys/BAX I187C. M and D indicate the monomeric and dimeric forms of each protein, respectively. The cross-linked VDAC2–BAX complex is indicated by a red arrow.

Molecular dynamics simulations and electrophysiology reveal stable α9-dependent pore occlusion by BAX.

A. Representative snapshots from coarse-grained MD simulations (Movies 1-3) illustrating different conformations of the VDAC2–BAX complex and the relative positioning of the soluble BAX domain with respect to the VDAC2 pore. VDAC2 is colored in grey, BAX in pink with the trigger site (orange), canonical groove (yellow), BH3 domain (red), and C-terminal α9 helix (blue). B. Estimated pore opening probability for the VDAC-BAX complex during three independent 10-µs MD simulations. For comparison, pore opening probability was recalculated from the trajectory shown in A after computational removal of BAX from the simulation (left). Grey bars are the conductance for each snapshot. The continuous green line is the floating average over 50 ns. Dotted line is the average of computed conductance of open pore probability over the whole simulation. C. D. E. are representative current recordings from a single VDAC channel before (left) and after (right) addition of 60 nM recombinant full-length monomeric BAX to the cis side of the membrane C. VDAC2 ; D. VDAC1 ; E. VDAC2 with BAX Δα9. Green dotted lines show VDAC-characteristing gating events28.Yellow arrows indicate the characteristic short-lived open-conductance substates of VDAC229. Applied voltages are shown in grey and the grey dashed line indicates the zero-current level. Stable current reduction was observed only after application of a voltage stimulation in the form of a slow triangular voltage ramp (5 mHz, ±60 mV) for at least 30 min (SFig. S2A). Current traces were digitally filtered at 500 Hz using an 8-pole low pass Bessel filter. Experiments were performed with VDAC inserted in a soybean polar lipid extract (PLE) membrane with bathing solutions of 150 mM KCl buffered with 5 mM HEPES at pH 7.4.

Integrative structural analysis reveals a membrane-inserted primed conformation of BAX bound to VDAC2.

A. Accessibility of cysteine residues in soluble BAX and VDAC2-bound BAX probed by NEM-PEG labeling. Single-cysteine BAX variants (C122, green; C62, black; C177, magenta), shown on the structure below, were produced by cell-free expression in the presence of preformed WT VDAC2- containing nanodiscs, purified by affinity chromatography, and labeled with NEM-PEG (5 kDa). Labeling was detected by western blotting using the pan-BAX antibody 2D2. PEGylated species, corresponding to solvent-accessible cysteines, display reduced electrophoretic mobility. Sol: soluble BAX produced in the absence of VDAC2. NR: non-retained fraction after Ni-NTA purification of BAX produced in the presence of 6His-VDAC2 nanodiscs. Comp: BAX in complex with VDAC2 nanodiscs, eluted with imidazole. B. Model of the VDAC2-BAX complex showing the positions of the three cysteines used in the NEM- PEG assay. VDAC2 is shown in grey and BAX in pink, with the canonical groove (yellow), BH3 domain (red), and C-terminal α9 helix (blue). The Cα atoms of the engineered cysteines are shown as spheres. In the inset, intermolecular cross-links identified by DSS cross-linking coupled to mass spectrometry (XL-MS) are highlighted in red. XL-MS identified contacts between the β6–β7 region of VDAC2 (Lys120 and Lys121) and the BH3 domain of BAX (Lys58 and Lys64). Reactive lysines are shown as spheres. C. Co-immunoprecipitation of WT BAX and 6His-VDAC2 following cell-free expression in the presence of VDAC2-containing nanodiscs using the pan-BAX antibody 2D2, the activation-specific antibody 6A7, or an anti-His antibody recognizing VDAC2. Resin without antibody was used as a negative control. D. Docking of the 6A7 Fab onto representative conformations of the VDAC2–BAX complex identifies models compatible with antibody binding and provides an additional structural constraint for refinement of the integrative model (SFig. 4). VDAC2 is shown in grey and BAX in pink, with the 6A7 epitope as green spheres, the canonical groove (yellow), BH3 domain (red), and C-terminal α9 helix (blue).

Structural model and functional role of the VDAC2–BAX complex.

A. Integrative structural model of the VDAC2–BAX complex using MD simulations, cysteine crosslinking, XL-MS, cryo-EM, electrophysiology, mutagenesis, and antibody accessibility measurements. VDAC2 is shown in grey and BAX in pink, with the 6A7 epitope as green spheres, the canonical groove (yellow), BH3 domain (red), and C-terminal α9 helix (blue). VDAC2 residues previously implicated in VDAC2 regulation of BAX (A171)17 or BAK (T167 and D169)19 are highlighted as red spheres. B. Proposed role of VDAC2 during BAX activation. Following membrane engagement and α9 insertion, VDAC2 captures and stabilizes a membrane-inserted, activation-competent BAX state characterized by BH3 exposure and accessibility of the 6A7 epitope while remaining pre-oligomeric. This intermediate may subsequently progress toward BAX oligomerization and apoptotic pore formation or undergo deactivation and retrotranslocation, possibly with a BCl2-family anti-apoptotic47.