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Summary BAX protein plays a key role in the mitochondria-mediated apoptosis. However, it remains unclear by what mechanism BAX is triggered to initiate apoptosis. Here, we reveal the mechanism using electron spin resonance (ESR) techniques. An inactive BAX monomer was found to exhibit conformational heterogeneity and exist at equilibrium in two conformations, one of which has never been reported. We show that upon apoptotic stimulus by BH3-only peptides, BAX can be induced to convert into either a ligand-bound monomer or an oligomer through a conformational selection mechanism. The kinetics of reaction is studied by means of time-resolved ESR, allowing a direct in situ observation for the transformation of BAX from the native to the bound states. In vitro mitochondrial assays provide further discrimination between the proposed BAX states, thereby revealing a population-shift allosteric mechanism in the process. BAX’s apoptotic function is shown to critically depend on excursions between different structural conformations. [ABSTRACT FROM AUTHOR]

BAX activation is a crucial step for commitment to apoptosis. Several activators, such as BimBH3-based therapeutic peptides and cleaved Bid (cBid) protein, can trigger BAX-mediated apoptosis, but it is unclear whether they proceed through the same pathway. Here we utilize PEGylation-based approach, which is shown to efficiently shield individual binding grooves in BAX from activators, to investigate and reveal that the activators take different routes to induce BAX-mediated apoptosis. Various spectroscopic/biochemical tools, including electron spin resonance, circular dichroism, fluorescence recovery after photobleaching, and label-transfer assay, were employed to reveal details in the processes. We observe a key mutant BAX 164-PEG that acts differently in response to cBid and BimBH3 stimuli. While BimBH3 directly interacts with the trigger groove (TG) to induce the conformational changes in BAX that includes the release of α9 from the canonical groove (CG) and oligomerization, cBid engages with CG and works with mitochondrial lipids to fully activate BAX. PEGylation-based approach is proven useful to shield individual binding grooves of BAX from apoptotic stimuli. Groove engagement in CG of BAX is required for a full cBid-induced BAX activation. This study has identified differences in the pathways involved during the initiation of BAX activation by full-length cBid protein versus synthetic BimBH3-based peptides. Our finding is potentially valuable for therapeutic application as the pore-forming activity of 164-PEG is independent from the cBid-mediated apoptotic pathways, but can be administrated by the synthetic short peptides. Unlabelled Image • A full BAX activation for MOMP proceeds with two steps essentially. • Accessibility of trigger groove of BAX is required for initiation of activation. • BimBH3 peptide and cBid take different routes to activate BAX. • Cleaved Bid interacts with the canonical groove to promote BAX activation. • Accessibility of canonical groove is not strictly required for BAX activation. [ABSTRACT FROM AUTHOR]