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

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BAX is a major effector of mitochondrial apoptosis and is activated through a series of conformational transitions that lead to mitochondrial outer membrane permeabilization. Genetic studies have established VDAC2 as an essential regulator of BAX-mediated apoptosis, yet the molecular basis of this regulation remains unresolved. The absence of direct structural and biochemical characterization of VDAC2–BAX interactions has prevented mechanistic understanding of how VDAC2 influences BAX activation. Here, using complementary biochemical, biophysical and structural approaches, we reconstituted and characterized a stable VDAC2–BAX complex. Notably, complex formation was specific to VDAC2 and was not observed with VDAC1, highlighting an isoform-specific role in BAX regulation. We show that VDAC2 captures and stabilizes a primed BAX conformation characterized by membrane insertion, exposure of the BH3 domain, and increased accessibility of the N-terminal activation region. By integrating AlphaFold3 predictions with biochemical, biophysical and structural constraints, we derive an experimentally supported structural model in which BAX is anchored through its α9 helix while its soluble domain partially extends over the VDAC2 pore. Together, our findings support a model in which VDAC2 stabilizes a membrane-inserted, activation-competent BAX intermediate. Rather than serving as a structural component of apoptotic pores, VDAC2 acts as a checkpoint that regulates progression along the BAX activation pathway. These results provide a molecular explanation for the long-recognized requirement of VDAC2 in mitochondrial apoptosis and establish an experimentally supported structural framework for a previously inaccessible intermediate in the BAX activation pathway.