VDAC2 stabilizes a membrane-inserted, primed intermediate of BAX activation

Reviewer #1 (Public review):

Summary:

The authors elegantly demonstrate a biochemically reconstituted approach to showcase the VDAC2-BAX interaction using lipid nanodiscs. The reconstitution method is specific to VDAC2 (and not VDAC1) and can capture several structural conformations. The authors show that the VDAC2-BAX heterodimer is sufficient for the direct capture and stabilization of BAX on the outer mitochondrial membrane by VDAC2. Their structural model demonstrates that a GXXXA motif within α-helix 9 of BAX drives its interaction with the β-barrel interface of VDAC2 in the membrane. AlphaFold 3 models suggest BAX adopts several distinct conformations, notably including both a strongly pore-occluding state and a loosely pore-occluding state. Functionally, electrophysiology experiments suggest that the addition of BAX modulates the voltage-gating function of VDAC2 by reducing conductance. Finally, conformation-specific antibodies and cross-linking mass spectrometry capture these structural rearrangements of BAX, reinforcing the proposed structural model.

Strengths:

Overall, the manuscript provides a solid structural and molecular rationale for BAX recruitment to VDAC2 and its subsequent oligomerization.

Weaknesses:

The authors have not sufficiently discussed key protein modifications during apoptosis in detail, especially regarding residues implicated in phosphorylation and their impact on VDAC2 association.

Overall, the manuscript is well written and presents an elegant biochemical and biophysical approach to identifying key functional states of the VDAC2-BAX complex. However, certain key functions of the complex are not extensively discussed or accounted for in the final model. For instance, components of this complex are phosphorylated in response to apoptotic or anti-apoptotic cues. Specifically, phosphorylation of S184 (located within the critical α-helix 9), T167, and S163 has key functions in promoting or preventing outer mitochondrial membrane translocation. How do the authors reconcile their structural models with the functional states of the complex generated in response to these signaling cues? This is particularly relevant given that the expression systems used here presumably yield proteins lacking these post-translational modifications (PTMs). The authors should consider running AlphaFold 3 predictions that incorporate key PTMs and discuss their potential functional impact. In its current state, the manuscript implies that unmodified BAX is sufficient for membrane translocation. Clarifying how PTMs influence pore occlusion and 6A7 epitope accessibility would significantly enrich this body of work.

Reviewer #2 (Public review):

In this study, the authors aimed to elucidate the precise molecular details underlying the BAX-VDAC2 interaction and subsequent BAX activation. To achieve this, they successfully combined AlphaFold3 structural modeling, cross-linking mass spectrometry, site-directed mutational screening, biochemical assays, and functional electrophysiology experiments.

The authors demonstrate that the direct interaction between BAX and VDAC2 is fully autonomous, occurring independently of any additional mitochondrial or cellular proteins. Their findings suggest that BAX exists on the outer mitochondrial membrane in two distinct populations: loosely membrane-associated, and tightly stabilized via its specific interaction with VDAC2. Crucially, the data overturn historical assumptions by demonstrating that BAX does not insert into the internal VDAC2 channel pore. Instead, the BAX α9 helix docks onto the lipid-facing outer surface of the VDAC2 β-barrel.

Interestingly, while the anchor is external, the soluble domain of BAX physically blocks the pore opening, leading to the observed occlusions of the VDAC channel. Following this docking event, the N-terminal 6A7 epitope of BAX becomes exposed, signaling a conformationally active state. However, the authors show that this structural activation does not trigger an immediate release from VDAC2 or prompt immediate oligomerization. Rather, BAX is maintained in a pre-oligomeric, primed intermediate state while bound to VDAC2. What ultimately regulates the release of this primed intermediate from VDAC2 to allow full oligomerization and pore formation remains an open question.

Altogether, this study provides pivotal mechanistic insights, clearly defining VDAC2 as a key checkpoint regulator of mitochondrial apoptosis.

Reviewer #3 (Public review):

Summary:

The authors are trying to provide the molecular basis for the emerging role of VDAC2 in mitochondrial apoptosis. They use multiple approaches from biochemistry, cell biology, and structural biology.

Strengths:

Isolating the VDAC2-BAX complex and providing the molecular basis of this interaction in mitochondrial apoptosis is pretty innovative and significant.

The authors have tried to validate their results using multiple approaches, which corroborates the quality of the study.

Weaknesses:

The scientific data and its presentation could be improved.