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Mitochondria maintain a biochemical environment that cooperates with BH3–only proteins (e.g., BIM) to potentiate BAX activation, the key event to initiate physiological and pharmacological forms of apoptosis. The sphingosine-1-phosphate metabolite 2-trans-hexadecenal (2t–hexadecenal) is one such component described to support BAX activation, but molecular mechanisms remain largely unknown. Here, we utilize complementary biochemical and biophysical techniques to reveal that 2t-hexadecenal non-covalently interacts with BAX, and cooperates with BIM to stimulate early-activation steps of monomeric BAX. Integrated structural and computational approaches reveal 2t–hexadecenal binds an undefined region – a hydrophobic cavity formed by core-facing residues of α5, α6, and gated by α8 – we now term the “BAX actuating funnel” (BAF). We define alkenal length and α8 mobility as critical determinants for 2t–hexadecenal synergy with BIM and BAX, and demonstrate that proline 168 allosterically regulates BAF function. Collectively, this work imparts detailed molecular insights advancing our fundamental knowledge of BAX regulation and identifies a regulatory region with implications for biological and therapeutic opportunities.