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

Abstract

BAX, a major effector of mitochondrial apoptosis, 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 that was not observed with VDAC1, highlighting an isoform-specific role in BAX regulation. We show that VDAC2 captures and stabilizes a primed BAX conformation displaying the hallmarks of activation, including membrane insertion, BH3 exposure, and increased accessibility of the N-terminal activation region. By integrating AlphaFold3 predictions with molecular dynamics simulations, biochemical, biophysical and structural constraints, we derive an experimentally-supported structural model in which BAX is anchored through its 9 helix while its soluble domain partially extends over the VDAC2 pore. Together, our findings support a model in which VDAC2 facilitates BAX membrane insertion and stabilizes a membrane-inserted, activation- competent BAX intermediate. Rather than serving as a structural component of apoptotic pores, VDAC2 acts as a regulatory checkpoint in BAX activation. These results provide a molecular explanation for the emerging role of VDAC2 in mitochondrial apoptosis and establish structural basis for a previously inaccessible intermediate in the BAX activation pathway.