A gated hydrophobic funnel within BAX binds bioactive lipids to potentiate pro-apoptotic function.

Mitochondria maintain a distinct biochemical environment that cooperates with pro-apoptotic BAX and BH3‑only proteins (e.g., BIM) to promote mitochondrial outer membrane permeabilization (MOMP), the key event to initiate physiological and pharmacological forms of apoptosis. The sphingosine-1-phosphate metabolite 2-trans-hexadecenal (2t‑hexadecenal) is a bioactive lipid that supports BAX-dependent MOMP. Using integrated structural and computational approaches, we determine that 2t‑hexadecenal binds within a distinct, dynamic region-a hydrophobic cavity formed by core-facing residues of α5, α6, and gated by α8-we now term the "BAX actuating funnel" (BAF). Complementary biochemical and biophysical techniques reveal that 2t-hexadecenal non-covalently interacts with the BAF and cooperates with BIM to stimulate intramolecular activation of monomeric BAX prior to membrane association. BAX α8 mobility and proline 168-mediated allostery are critical determinants for 2t-hexadecenal synergy with BAX and BIM, as is alkenal length to stimulate BAF function. Collectively, this work imparts detailed molecular insights into how pro-apoptotic BCL-2 proteins and bioactive lipids non-covalently cooperate to initiate the mitochondrial pathway of apoptosis with implications for biological and therapeutic regulation.