BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Because of its role as a critical gatekeeper of cell death, pharmacologic modulation of BAX has the potential to respectively activate or inhibit apoptosis in hematologic diseases characterized by unrestrained cell survival or pathologic cell death. BAX activation is believed to be a highly regulated, multi-step process involving an interaction-triggered conformational change, mitochondrial translocation, and oligomerization that ultimately leads to mitochondrial dysfunction and apoptosis. Anti-apoptotic proteins, such as BCL-2 and BCL-XL, counteract BAX-mediated apoptosis. Although an interaction site that endows anti-apoptotic proteins with survival functionality has been defined and drugged, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that target BCL-2 family proteins in situ, directly initiate BAX-mediated mitochondrial apoptosis in vitro, and suppress leukemia in vivo. Here we demonstrate by NMR analysis that a BIM SAHB peptide binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. In five distinct assays that measure ligand-induced BAX activation, BIM SAHB directly and dose-responsively triggers BAX. The specificity of the BAX-BIM SAHB interaction is highlighted by point mutagenesis that abrogates functional activity in vitro and in cells, confirming that BAX activation is initiated at this novel structural location. Thus, we report the first structural analysis of a BH3 death domain alpha-helix bound to a full length pro-apoptotic multi- BH domain protein, unveiling a novel site of protein interaction for direct activation of BAX and pharmacologic modulation of apoptosis.

Disclosures: Walensky:Aileron Therapeutics, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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