Abstract 1383: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

Abstract 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-associated intramolecular rearrangements prior to membrane permeabilization. Integrated structural and computational approaches suggest 2t-hexadecenal binds an undefined region - a hydrophobic cavity formed by core-facing residues of α5, α6, and gated by α8 - we term the "BAX actuating funnel (BAF).” Alkenal length and α8 mobility are critical determinants for the interaction, intramolecular rearrangements, and functional outcome of 2t-hexadecenal binding to the BAF; we also present a first-in-class small molecule that functionally mimics 2t-hexadecenal contributions to apoptosis. Collectively, this work imparts detailed molecular insights into how pro-apoptotic BCL-2 proteins and lipids assemble to initiate the mitochondrial pathway of apoptosis, and reveals the therapeutic potential of targeting the BAF. Citation Format: Jerry Edward Chipuk. A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1383.