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

Domain: chemistry
Published: 2026-02-25
Editorial outcome: major_revision

Abstract
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.

Introduction
Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by engaging the BCL-2 family of proteins to induce BAX-dependent mitochondrial outer membrane permeabilization (MOMP) and apoptosis1. Despite sequence and structural similarity with multiple globular BCL-2 family proteins, BAX is distinct in that it converts from an inactive cytosolic monomer to a pore-forming oligomer at the outer mitochondrial membrane (OMM). Upon transient triggering with direct activator BH3‑only proteins (e.g., BIM, BID), BAX undergoes a series of intramolecular rearrangements and structural refoldings that ultimately result in translocation to the OMM, oligomerization, and MOMP2–7. An important structural event during this process is α9 helix mobilization from its residence within the BAX BC groove, which simultaneously supports OMM translocation, BAX:BAX interactions, and propagation of the activation process leading to proteolipid pore formation. We refer to this series of structural rearrangements as the BAX activation continuum, which is subdivided into an activation phase (triggering) and functionalization phase (pore formation)8.

There are two general requirements for potent BAX activation: protein-protein interactions to trigger the monomer, and protein-lipid interactions to initiate and stabilize refolding of BAX into multimeric conformers. These requirements are proposed to occur in distinct phases of the activation continuum, and most insights into protein-lipid interactions focus on how BAX interacts within the OMM to form pore-like structures9–12. This has led to a modern conceptualization in which mitochondrial membranes actively cooperate with the BCL‑2 family to control cell death commitment through the regulation of mitochondrial shape and curvature, dynamics machinery, lipid milieu, and intra-organellar communication13–15. Notably, mitochondrial endoplasmic reticulum contact sites (MERCS) maintain lipid homeostasis between the organelles and supply lipids that are critical for MOMP16.

One such example is the terminal end-product of the sphingolipid pathway 2-trans-hexadecenal (2t‑hexadecenal)—which is required for BAX-mediated MOMP following triggering by BIM or BID17. 2t‑hexadecenal is formed by the irreversible cleavage of sphingosine-1-phosphate (S1P) and is further metabolized to hexadecenoyl‑CoA and palmitoyl-CoA for fatty acid synthesis pathways18,19. The resident enzymes responsible for generating and catabolizing 2t‑hexadecenal are enriched at MERCS, and genetically modulating 2t‑hexadecenal levels alters sensitivity to human BAX in yeast20. Previously, the requirement for 2t‑hexadecenal in BAX-mediated MOMP was described and subsequent investigations have proposed a covalent mechanism by which 2t‑hexadecenal alkylates BAX at the membrane17,21,22. However, the non-covalent role of 2t‑hexadecenal to cooperate with BH3-only proteins and promote BAX activation prior to membrane association remains an unanswered question proposed by the original investigation.

Here, we utilize biochemical, biophysical, structural, and computational approaches to systematically demonstrate that 2t‑hexadecenal directly actuates BAX through non-covalent interactions at a previously undefined region—a funnel-shaped hydrophobic cavity formed by core-facing residues of α5, α6, and gated by α8, which we term the BAX Actuating Funnel, or BAF. Our results suggest that BIM-mediated BAX triggering mobilizes α8, making the BAF accessible, and that binding of 2t‑hexadecenal promotes BAX functionalization. Furthermore, we identify chemical and structural determinants underlying 2t‑hexadecenal:BAX interactions and reveal that mutation of proline 168 in the loop between α8 and α9 allosterically deforms the BAF and subsequently disrupts the function of 2t‑hexadecenal. Collectively, this model advances our understanding of BAX structure-function relationships by characterizing a non-covalent protein-lipid interaction responsible for stimulating monomeric BAX activation and identifies a previously under-appreciated regulatory domain for both cell biology and therapeutic investigations.

Results
2t‑hexadecenal directly activates BAX through non-covalent interactions

Previous work demonstrated that 2t‑hexadecenal (henceforth, hexadecenal) was required for potent BAX-mediated MOMP and that sphingolipid precursors are supplied to mitochondria via interactions with heterotypic membranes17. To assess whether ectopic hexadecenal exposure could engage BAX activation in cellulo, we treated SV40-transformed mouse embryonic fibroblasts (MEFs) with increasing concentrations of hexadecenal and measured the apoptotic response using our real-time multi-visitation microscopy technique, SPARKL23. Ectopic hexadecenal (“2t16”) did not induce cell death until high concentrations were utilized (Figs. 1A and S1A). We reasoned that any pro-apoptotic signaling may have been mitigated by the repertoire of anti-apoptotic BCL‑2 family proteins, and indeed, co-treatment with the BH3-mimetic ABT‑737 revealed an apoptotic phenotype in response to ectopic hexadecenal (Fig. 1A). To determine if the apoptotic response required triggering by direct activators, we utilized Bim−/−Bid−/− double knockout (DKO) MEFs and observed a loss of sensitivity to hexadecenal at lower concentrations co-treated with ABT‑737, suggesting that BAX activation by direct activators was part of the underlying mechanism (Fig. 1B). Finally, we replicated this experiment in Bax−/−Bak−/− DKO MEFs and observed no cell death in response to hexadecenal indicating a requirement for the effector proteins (Fig. 1C). Of note, high-concentration hexadecenal exhibited apoptosis as a single treatment and in conjunction with ABT‑737 in both wild type (WT) and Bim−/−Bid−/− DKO MEFs, but not in Bax−/−Bak−/− DKO MEFs, potentially indicating that this concentration of hexadecenal was directly activating BAX. Collectively, these data may suggest that hexadecenal induces apoptotic cell death by acting on effector BCL‑2 family proteins.

There are reports that ectopic hexadecenal can form adducts with DNA and generate oxidative stress resulting in apoptosis, and we could not eliminate the possibility that ectopic hexadecenal was targeting additional substrates or stress pathways24,25. Therefore, we interrogated whether hexadecenal directly promoted BAX-mediated pore formation by utilizing recombinant BAX protein and large unilamellar vesicles (LUVs), which are biochemically-defined liposomes that mimic the major lipid composition of the OMM9, and assessed BAX activation by measuring LUV permeabilization. While recent studies have incorporated hexadecenal directly into the formulation of LUVs22, we aimed to investigate hexadecenal-mediated BAX activation without altering the biochemistry of the LUV membrane. BAX treated with hexadecenal demonstrated dose-dependent activation and LUV permeabilization (Fig. S1B). Importantly, ectopic hexadecenal alone did not disrupt or cause leakage of LUVs (Fig. S1C). In the cell, BAX activation is mediated primarily through BCL‑2 family direct activators predominantly, BIM26—and we had observed apoptotic resistance in the Bim−/−Bid−/− MEFs; therefore, we sought to assess the cooperation of BIM and hexadecenal on BAX-mediated membrane permeabilization. We treated BAX with a range of activating concentrations of BIM‑BH3 peptide and observed a dose-dependent increase and acceleration in LUV permeabilization in response to hexadecenal (Figs. 1D, F and S1D). Furthermore, we determined that the additional response to BIM‑BH3 and hexadecenal was synergistic (Fig. 1G). Previous work indicated that the saturated form of 2t‑hexadecenal hexadecanal (“16CHO”)—did not induce BAX oligomers in cross-linking studies17, and indeed, we did not observe BAX-mediated pore formation or synergy with BIM in response to hexadecanal (Figs. 1H–I and S1E). Similarly, we modified hexadecenal by reacting it with a hydrazide compound and determined that the resulting molecule (“2t16‑NN‑EtOH”) did not promote BAX activation or synergize with BIM stimulation (Figs. 1J–K and S1F). These data indicate that hexadecenal promotes BAX pore formation and synergizes with BIM-mediated activation. Of note, however, we cannot exclude the possibility that the alteration of the aldehyde moiety in 2t16‑NN‑EtOH may have compromised the reactivity of the conserved double bond.

Despite treating BAX with hexadecenal directly, we could not entirely rule out the possibility that increased permeabilization could have been due to the lipidic aldehyde interacting with LUVs and resulting in a more permissive environment for BAX pore formation. Therefore, we utilized microscale thermophoresis (MST) to determine whether hexadecenal directly bound to BAX and observed a dose-dependent shift indicating changes to the molecular volume of BAX in response to hexadecenal (Fig. 2A). Of note, the MST data determined an EC50 value of 0.182 μM, or when hexadecenal was ~180-fold the concentration of BAX, which is approximately the ratio that exhibited the greatest synergy with BIM-mediated BAX membrane permeabilization (Figs. 1G and S2A). As such, we utilized a range of hexadecenal concentrations that represented ratios aligning with the MST data in our subsequent assays. Interestingly, the saturated hexadecanal aldehyde did not induce a similar change in BAX thermophoresis, suggesting that loss of the α,β double bond disrupted interaction with BAX and subsequent activation (Figs. 2B and S2B).

Hexadecenal is an α,β-unsaturated aldehyde and is capable of modifying nucleophiles (e.g., cysteine residues) through Michael addition, and the loss-of-function of the saturated aldehyde and 2t16‑NN‑EtOH, could have suggested a chemical reaction mechanism27. Indeed, recent publications have reported that hexadecenal covalently modifies over 500 cellular proteins, including BAX, though the studies disagree on which cysteine residue was modified21,22. To determine if BAX was being covalently modified within our assays, we incubated BAX with hexadecenal, subjected the sample to proteolytic cleavage, and analyzed the peptide fragments by tandem liquid chromatography-mass spectrometry (LC-MS). We did not observe alkylation by hexadecenal (m+238.229 Da) on either of the BAX cysteine residues (C62, C126); as a control, we did detect cysteines modified by the alkylating agent iodoacetamide (m+57.021 Da) (Fig. S2C). However, we could not eliminate the possibility that hexadecenal-modified BAX may have not been detected due to limitations of the digestion or peptide detection; therefore, we corroborated our results using native intact protein time-of-flight (TOF) mass spectrometry. We utilized an oligomerization-deficient BAX mutant (BAXG108V) to prevent BAX aggregation and observed no mass shift in response to hexadecenal, though we did observe modification by iodoacetamide (Fig. 2C). Additionally, we replicated this experiment using an alternative oligomerization-deficient BAX mutant (BAXR109D) to confirm that these results were not specific to either mutation (Fig. 2D).

To decisively conclude that the mechanism of hexadecenal synergy was independent of cysteine modification, we replicated our LUV permeabilization studies using cysteine-deficient BAX generated by two complementary approaches. First, we functionally inhibited the reactivity of the BAX cysteines via iodoacetamide capping and demonstrated that there was no loss of activation or synergy in response to BIM‑BH3 and hexadecenal (Fig. 2E−G). Next, we utilized a cysteine-replacement BAX mutant (BAXC62S, C126S, “BAX2S”), which exhibited no changes to stability or melting temperature (Fig. S2D). Compared to wild-type BAX (“BAXWT”), BAX2S remained sensitive to hexadecenal-mediated changes in melting temperature, pore formation, and synergy with BIM‑BH3 peptide (Figs. 1D–G, 2H–I, and S2E−G). Additionally, BAX2S remained similarly insensitive to the saturated hexadecanal aldehyde (Fig. S2H–I). Collectively, these results indicate that there is a non-covalent mechanism by which hexadecenal promotes BIM-mediated BAX activation through a direct interaction.

2t‑hexadecenal cooperates with BIM to promote early-activation of monomeric BAX

The BAX activation continuum can be divided into distinct phases of activation and functionalization: the cytosolic monomer gets activated and undergoes intramolecular rearrangements and conformational changes that result in translocation to the OMM; subsequently, active BAX proteins in the OMM undergo large-scale conformational changes, oligomerize, and mature into pore-forming units. Our data thus far have measured BAX functionalization (i.e., pore formation) and demonstrated that hexadecenal synergizes with BIM to promote membrane permeabilization. However, we utilized a direct treatment model of hexadecenal and BAX protein (compared to membrane incorporation), indicating that the mechanism of action likely occurs on BAX found in the cytosol and prior to interactions with the OMM.

To investigate the effect of hexadecenal specifically on the activation phase of BAX, we utilized a technique we developed called FLAMBE, which monitors activation-induced intramolecular rearrangements within BAX (i.e., rearrangements that result in early-activation structural hallmarks such as displacement of the α1–α2 loop and mobilization of the C-terminal α9 helix)4,8. FLAMBE observes real-time early-activation of BAX by measuring changes in Polarization resulting from the kinetic binding and dissociation of a TAMRA-labeled BAK‑BH3 peptide (BAKTAMRA); broadly, BAX activation can be inferred by measuring a reduction of Polarization over time (Fig. S3A). Kinetic FLAMBE data can further be parameterized into time-of-maximum-Polarization (Tmax) and endpoint Polarization (EP) for trend overviews and comparative analyses between treatment conditions (Fig. S3B). As an example, BAX treated with a range of BIM concentrations exhibits a dose-dependent pattern of BAKTAMRA dissociation (measured as a reduction in Polarization over time) indicative of BAX activation (Fig. S3C).

Using FLAMBE, we observed BAXWT activation in the presence of hexadecenal as demonstrated by a reduction in Polarization over time, indicating that high concentrations of hexadecenal could induce a “direct-activator”-like effect on BAX monomers, which aligns with the LUV permeabilization data (Fig. 3A). To confirm that this phenotype was not due to covalent modification of cysteine residues, we replicated the FLAMBE experiment with BAX2S and observed the same activation profile (Fig. 3B). By contrast, neither BAXWT nor BAX2S activated in response to the saturated hexadecanal aldehyde, though we did observe some destabilization of the BAX:BAKTAMRA heterodimer at the higher concentrations (Fig. S3D–E). We hypothesized that hexadecenal cooperates with direct activators instead of activating BAX de novo within the cell, and thus we investigated whether the synergy between hexadecenal and BIM occurs during the early-activation phase. We treated BAX2S with a non-activating concentration of BIM‑BH3 to generate a population of stable BIM:BAX:BAKTAMRA heterotrimers (Fig. 3C, yellow data). The addition of a non-activating concentration of hexadecenal resulted in activation of the primed BAX2S population (Fig. 3C, left panel); moreover, activation of primed BAX2S was observed with several non-activating concentrations of hexadecenal (Fig. 3C, right panel). When we induced a “triggered” BAX population (i.e., mildly activated by BIM), we similarly observed increased activation by non-activating concentrations of hexadecenal, as measured by increased kinetics of BAKTAMRA dissociation, greater shifts in parameterized metrics, and reduction in area under the curve (Figs. 3D and S3F). Importantly, we observed the same outcomes with BAXWT, confirming that the mechanism was not due to the endogenous cysteines or their mutation (Fig. S3G). Critically, we observed no disruption of a BCL‑xL/BAKTAMRA complex at comparative hexadecenal concentrations indicating that dissociation of BAKTAMRA from BAX was due to BAX activation and not competition for the BC groove or a non-specific detergent-like effect (Fig. S3H).

The consequence of BAX activation is translocation to the OMM and oligomerization, and while it is largely agreed that high-molecular weight oligomers are formed within the OMM, there is evidence supporting that physiologically-activated BAX forms low-order multimers (e.g., dimers) in solution prior to integrating with membranes6,8,28. We investigated the consequence of hexadecenal-mediated synergy with BIM-activated BAX by performing size exclusion chromatography (SEC) and observed a substantial shift from monomeric to dimeric BAX when co-treated with BIM‑BH3 peptide and hexadecenal (Fig. 3E). To confirm that the earlier peak was BAX, we treated fluorescently-labeled BAX, subjected it to SEC, and screened the fractions for fluorescence. The monomeric BAX peak (fractions 28–31) shifted slightly left upon addition of hexadecenal or BIM‑BH3 peptide (fractions 27–30), likely due to binding-dependent changes in molecular volume, and dimeric BAX species were observed in the BIM-treated sample (fractions 23–26); the co-treatment of hexadecenal and BIM‑BH3 resulted in an increased shift and intensity indicating a greater percent of the BAX population formed multimeric species (fractions 22–26) (Fig. 3F). By contrast, no shift in the monomeric peak or BIM-induced dimer peak was observed with hexadecanal (Fig. 3G). Of note, BAX activated in solution does not readily form high-molecular weight species without the stabilizing and concentrating influence of a hydrophobic environment (e.g., a membrane or micellar detergent)8,29; as a reference, we were able to observe BAX oligomers generated with a detergent (BAXO)30, and we interpret these results to indicate that hexadecenal is not exhibiting a detergent-like effect on BAX. These data collectively demonstrate that hexadecenal promotes monomeric BAX activation downstream of BCL‑2 protein interactions, following activation by direct activators, and before interactions with a membrane.

An alpha8 helix-gated funnel-shaped hydrophobic cavity in the BAX core interacts with 2t‑hexadecenal

Hexadecenal synergized with BIM-mediated BAX activation and we observed no evidence of competition with the BAKTAMRA peptide in our FLAMBE assays, suggesting that hexadecenal was binding to a site distinct from either the trigger site or BC groove—the two BH3-interacting sites, respectively31,32. Despite having a relatively smooth surface and no obvious “binding pocket,” several studies have identified small molecules that bind to BAX, either at BH3-interacting sites or allosterically33–35. To identify putative interaction sites, we performed 2D 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) of 15N-labeled BAXWT treated with hexadecenal and measured a multitude of peak shifts (Fig. S4A−C). Several residues exhibited significant chemical shift perturbations (CSPs) in response to hexadecenal, some of which were within unstructured regions (such as the N-terminus) or highly exposed areas (such as α4 and α9), but notably the two cysteine residues (C62 and C126) did not reach significance (Fig. 4A). Interestingly, several shifts were observed in core-facing residues of α2, α5, α6, and α8, as well as bulky residues proximal to α8 within the α4–5 loop and α7. Additionally, several residues in this region exhibited altered resonance morphology, such as peak splitting or merging (Fig. 4A, right). Critically, we did not observe any global loss of signal or peak intensities suggestive of protein loss due to either activation-induced oligomerization or degradation. To determine whether the shifted residues were specific to the activation mechanism, we compared this CSP profile against CSPs of BAXWT treated with the non-activating saturated aldehyde hexadecanal and observed that residues exhibiting significant shifts were more localized to accessible regions of BAX along α2, α3, and α9 (Fig. S4D). Comparing the CSPs of the two aldehydes highlighted that many of the hexadecenal-induced shifts were not observed with the saturated aldehyde (Fig. S4E). These core-facing residues are not believed to be readily accessible and therefore were likely to be meaningful for the mechanism of hexadecenal interaction and function.

Inspection of the BAX structure revealed a cavity formed by hydrophobic residues in the core of the protein. The cavity shape resembles a funnel and is comprised of two topographies: a wide and shallow “mouth” formed by residues in α1, α2, α5, and α8; a narrow “neck” extending into the BAX core between α1, α5, and α6 (Fig. 4B). Of note, the cavity is conserved in each of the structures of the NMR ensemble, albeit with differing shapes and connectivity, suggesting that it is a highly dynamic feature of monomeric BAX (Fig. S4F). We hypothesized that this hydrophobic “funnel” could be a desirable interaction site for hexadecenal—an inherently lipidic aldehyde—and therefore we performed unconstrained in silico docking calculations using the SwissDock web service to model interactions with BAX. Binding modalities of hexadecenal were clustered into a few distinct regions on BAX, but 59.4% of the poses were proximal to α8, aligning with the CSPs observed by NMR (Fig. 4C).

Our functional interrogations indicated that hexadecenal synergizes with BIM-mediated BAX activation, and we reasoned that BIM-induced intramolecular arrangements may induce flexibility and/or mobility of α8, making the funnel accessible to hexadecenal. SwissDock utilizes rigid receptor docking, which results in the α8 helix firmly blocking the funnel. To create a funnel-accessible structure, we removed α8 (BAXΔα8) and docking against the BAXΔα8 structure revealed a clear preference for the funnel with 97.3% of hexadecenal poses positioned within the funnel (Fig. 4D). Interestingly, when we examined the solution NMR structure of BAX bound to a stapled BIM‑BH331—a structure representing a quasi-active, “triggered” BAX—we observed an enlargement of the hydrophobic funnel, most notably in the neck of the funnel, and this was conserved across the structures within the ensemble (Figs. 4E and S4G). In the inactive BAX structure, the neck of the funnel is cinched into discontinuous cavities by residues of α5 and α6; in contrast, the BIM-bound active monomer structure has a connected cavity due to a shift in the residues lining the funnel, increasing both the depth and width. Docking hexadecenal against the BAX:BIM structure with α8 removed (BAXΔα8:BIM) revealed that the aldehyde was positioned within the funnel, frequently posed as inserted into the neck of the funnel (Fig. 4F).

To substantiate our in silico conclusions that hexadecenal interacts within this cavity, we engineered a structural mutant of BAX to restrict the mobility of α8 and the subsequent access to the hydrophobic funnel. We introduced two cysteine residues into BAX2S (L59C, C62S, C126S, L162C; “BAXα8”) that could be oxidized to induce a disulfide tether between α2 and α8 (Fig. 4G). We characterized the consequence of the new mutations by activating BAXα8 with BIM‑BH3 peptide and assessing LUV permeabilization. Reduced BAXα8 (i.e., “unlocked”) remained functional and exhibited BIM-induced LUV permeabilization (Fig. S4H); in contrast, oxidized BAXα8 (i.e., “locked”) demonstrated no permeabilization (Fig. S4I). Several studies have characterized that BAX functionalization and pore formation require large-scale conformational reorganization, which involves the separation of α8 from core helices, and therefore it is not surprising that locked BAXα8 was a “functionally dead” structural mutant5–7,36. However, we recently demonstrated that several historically “dead” BAX mutants remain sensitive to BH3-induced activation and demonstrate early-activation structural hallmarks despite not maturing into functional pore-forming conformations8. SEC experiments revealed that locked BAXα8 exhibited a shift in response to BIM‑BH3 but was unaffected by either hexadecenal alone or coupled with BIM-mediated activation (Fig. 4H). To further investigate whether the immobilized α8 helix would disrupt sensitivity to hexadecenal, we studied BAXα8 activation using our FLAMBE assay. Both the unlocked and locked forms of BAXα8 demonstrated activation by BIM‑BH3, indicating that the mutant still exhibits activation-induced intramolecular rearrangements (Fig. S4J–K). Critically, locked BAXα8 did not activate when treated with hexadecenal (Fig. S4L). Moreover, BIM-primed BAXα8 displayed no activation or synergy in the presence of hexadecenal (Figs. 4I and S4M). These data substantiate the results of our docking investigations and the conclusion that hexadecenal-induced BAX activation is mediated through non-covalent interactions with the hydrophobic funnel in the BAX core, which is exposed following BIM triggering and subsequent α8 mobilization. Given that this hydrophobic cavity potentiates BAX activation, we termed this site the “BAX Actuating Funnel”, or BAF.

Aldehyde length and BAF topography are determinants of BAX activation

Neither the saturated aldehyde hexadecanal nor the hydrazide-modified hexadecenal activated BAX suggesting that the mechanism is distinct to the structure of 2t‑hexadecenal. We next considered the relevance of aldehyde length on interactions with the BAF by utilizing a panel of 2t‑alkenals ranging from 5 to 13 carbon chains (henceforth, “alkenals”). HSQC experiments with a mid- or short-chain alkenal, 2t‑nonenal (“2t9”) and 2t‑pentenal (“2t5”), respectively, demonstrated CSP profiles that were similar to hexadecenal, but were also more diffuse across BAX residues (Fig. 5A). While there was some conservation of CSPs observed in residues within or proximal to α8, the shorter alkenals exhibited a plethora of shifts within α4 and α9 as well as a general trend of interacting with solvent-exposed residues (Fig. S5A). These data may also indicate that shorter alkenals are more promiscuous, as nonenal and especially pentenal interacted with several accessible regions of BAX, exhibited decreasing specificity at higher concentrations, and displayed weaker CSPs (Fig. S5B–C).

Docking investigations with alkenals ranging from 5 to 15 carbon chains supported this hypothesis by demonstrating a length-dependent specificity for the α8 region (in the full-length structure, BAXFL) or the BAF (in the BAXΔα8 structure) (Figs. 5B and S6A, B). When docked against the BAXΔα8:BIM structure, most alkenal species were predicted to interact with the larger BAF, though many were not positioned in the depth of the funnel and short-chain aldehydes could not occupy both the mouth and neck of the BAF in the same pose (Fig. S6C). In the BIM-bound BAX structure, mobilization of the α1–α2 loop created a region that was predicted to be an interaction site, and longer aldehyde structures were disproportionately positioned in this region; we believe this was an artifact of rigid receptor docking and not a physiological phenomenon, and therefore we did not quantify the percentage of binding poses localized to the BAF. The precursor of 2t‑hexadecenal, sphingosine 1-phosphate (S1P), was previously identified as a requirement for BAK-mediated MOMP but did not directly promote BAX-mediated MOMP17. We docked the structure of S1P against BAX models and observed a reduced preference for the BAF and the tail could not occupy the neck of the BAF when modeled against BAXΔα8:BIM structure, likely due to the bulky and negatively charged phosphate group (Figs. 5B and S6A–C). Collectively, NMR and unconstrained, global docking assessments supported our hypothesis that aldehyde length and structure promote specificity for the BAF.

Next, we tested our hypothesis by investigating which, if any, alkenals could phenocopy hexadecenal function and BAX activation. When subjected to MST, BAXWT exhibited altered thermophoresis with each of the alkenals; though the fit of the data demonstrated a trend in which the slope increased with aldehyde length (Figs. 5C and S7A). To interrogate the functional consequence of alkenals interacting with BAX, we next subjected each alkenal to FLAMBE analysis and observed a dependency on aldehyde length for BAX2S activation (Fig. 5D). Interestingly, at the concentrations we tested, 2t‑undecenal (“2t11”) and 2t‑dodecenal (“2t12”) displayed some disruption of BAX2S:BAKTAMRA interactions, similar to the results with hexadecanal, but BAKTAMRA was able to re-bind and did not exhibit an activation profile (Figs. S3E and S7B). When compared to hexadecenal, only 2t‑tridecenal (“2t13”) exhibited a clear dose-dependent activation trend, albeit less potently than hexadecenal (Figs. 5D–E and S7B). To definitively determine alkenal-induced BAX activation, we performed LUV permeabilization studies and confirmed a length-dependent sensitivity for BAX2S-mediated pore formation (Figs. 5F and S7C). Additionally, these results were conserved in experiments using BAXWT. These observations align with a prior study demonstrating that short-chain alkenals exhibited greater reactivity with BAX but were paradoxically less proficient at inducing BAX activation compared to long-chain alkenals22. It has been reported that 2t‑alkenals exhibit reduced reactivity in a length-dependent manner, despite having similar electrophilic indices, possibly due to differences in steric hinderance or relative solubility/hydrophobicity, though this was not specifically measured for 2t‑hexadecenal or long-chain alkenals37,38. Taken together with our structural and modeling data, these results indicate that chain length is a determinant of α,β-unsaturated alkenals to specifically target the BAF and activate BAX.

Having identified determinants of ligand specificity, we next sought to characterize the residues of the BAF that regulate interactions with, and sensitivity to, hexadecenal. We performed molecular docking of hexadecenal against the BAX:BIM structure using the Glide software with a docking grid centered on the BAF. As we expected, the residues lining the BAF that interact with hexadecenal were predominantly hydrophobic residues of α5 and α6 and all predicted poses were positioned into the neck of the BAF in a “tail-first” orientation (Fig. 6A, B). Compared against the docked position of the inactive 2t16‑NN‑EtOH molecule, the modified head group induced a pose that did not reside in the BAF (Fig. S8A). When we analyzed the top ten scored poses for 2t16‑NN‑EtOH, we observed several possible poses including the neck of the BAF, extensions towards α9, or entirely nested in the mouth of the BAF, indicating a departure from the residues predicted to stabilize the hexadecenal pose (Fig. S8B). Therefore, these in silico determinations suggest that 2t16‑NN‑EtOH fails to synergistically activate BAX due to altered binding conferred by the modified structure.

Interestingly, when we introduced additional positional constraints on the oxygen atom of hexadecenal, we were able to generate a “head-first” orientation, which did exhibit slightly better binding scores according to Glide; despite this, we could not replicate this orientation without dictating the position of the oxygen within the BAF. As such, we cannot decisively conclude which orientation may be preferred or representative of physiological interactions, but the conclusion that hexadecenal resides within the depth of the BAF is conserved in both models.

We next performed a virtual mutagenesis screen aimed at disrupting the BAF and identified that V110L, L113M, or L144F mutations were likely to alter the BAF (Fig. 6C). Indeed, performing molecular docking on these mutant structures demonstrated worse binding scores and altered hexadecenal positioning due to the disruption of BAF topography (Fig. 6D, E). Of note, we ran clustering analysis on all the docked poses for each mutant and found that the poses were sufficiently similar to be clustered together with the exception of BAXL113M, which still positioned one pose into the shallower BAF; however, this pose had worse scoring compared to the top L113M hits.

To corroborate our in silico conclusions, we generated recombinant protein for these BAF mutants and tested them for BAX functionality. Several established BAX point mutants result in a deficient or functionally dead protein, and so we first determined the melting temperature of our BAF mutants. Compared to WT, each BAF mutant exhibited a lower melting temperature, suggesting that these mutations would not be functionally deficient (Fig. 6F). While the lower melting temperature may also suggest reduced stability of the monomeric protein, we did not observe any increased redistribution to oligomeric species during protein purification and storage. Furthermore, we confirmed that each BAF mutant responded to BIM activation with similar kinetics and endpoint, noting only slight insensitivity of BAXL113M at low BIM concentrations but similar activation at high concentrations (Fig. S8C, D). Critically, each BAF mutant demonstrated complete insensitivity to hexadecenal and exhibited a loss of synergy with BIM-primed BAX (Fig. 6G, H and S8E, F). It is worth noting that BAXL113M did retain some synergy of BIM and hexadecenal, albeit substantially reduced, and we suggest that this may be a consequence of a shallower, but relatively intact, BAF in the BAXL113M structure (Fig. 6E). As an aside, we did generate and screen candidate mutations altering the bulky BAF residues (i.e., F114 and F143), but observed reduced stability, oligomerization during purification, and substantially increased sensitivity to BIM, which obfuscated interpretations with hexadecenal. Interestingly, a prior study also noted that mutation of F114 is highly sensitizing39, and we posit that substituting these bulky residues enlarges the BAF and subsequently destabilizes BAX (see Discussion). Collectively, these investigations indicate that the activating mechanism of hexadecenal is the ability of the aldehyde to reside in the depth of the BAF, and alteration to the BAF shape or aldehyde size disrupts this fundamental interaction.

Proline168 allosterically controls the BAF and 2t‑hexadecenal function is lost in the BAXP168G structure

Our modern understanding of the BAX structure-function relationship includes the concept of allostery, in which interactions with BAX can cause structural rearrangements in distal regions. This concept is observed during BIM-mediated activation, which elicits mobilization of α9 from the BC groove31, or binding of a sensitizing molecule to the hairpin pocket, which provokes exposure of the α1−2 loop34—both structural events are hallmarks of BAX activation4. Recently, the structure of BAX containing a mutation of proline 168 (BAXP168G) was proposed to cause rotation of bulky side chains in the distal α4−5 and α7−8 loops40. Despite identification of P168 as critical for BAX activation, translocation, and pore-forming ability over 20 years ago41, and its recent identification as an arising loss-of-function mutation conferring resistance to Venetoclax in acute myeloid leukemia42, a mechanistic explanation for its requirement has remained elusive. We hypothesized that the alternative rotamers in bulky α8-proximal residues may consequently alter the BAF and, indeed, the neck of the BAF is lost in the BAXP168G structure (Fig. 7A). Furthermore, molecular docking against the BAXP168G structure positioned hexadecenal with the carbon chain now pivoting towards α1 residues (Fig. 7A, B). Directly comparing between poses of hexadecenal docked to WT and P168G structures revealed distinct interaction signatures favoring interactions with α5/α6 or α1 residues, respectively (Fig. 7C).

We reasoned that disruption of the BAF in BAXP168G would inhibit interactions with hexadecenal. Using our FLAMBE assay, we demonstrated that BIM-primed BAXP168G did not activate in response to hexadecenal as compared to BAXWT (Figs. 7D and S9A). Furthermore, BAXP168G exhibited a reduced shift in melting temperature due to hexadecenal (Fig. 7E). As previously reported, BAXP168G was less sensitive to BIM activation and resulted in attenuated membrane permeabilization, but notably was not entirely functionally dead (Fig. S9B). In contrast, BAXP168G demonstrated no activation in response to hexadecenal and only minor synergy with BIM at activating concentrations, suggesting that activation-induced molecular rearrangements are the limiting factor for the synergistic function of hexadecenal (Figs. 7F, G and S9C).

As detailed by others40, we also observed a significantly higher melting temperature in BAXP168G compared to BAXWT, suggesting a stabilization of the monomeric conformer (Fig. 7E). Finally, we replicated our apoptosis experiments in Bax−/−Bak−/− DKO MEFs transduced to stably express either BAXWT or BAXP168G (Fig. S9D). The DKO MEFs reconstituted with BAXWT exhibited greater sensitivity to ectopic hexadecenal compared to BAXP168G-expressing MEFs (Fig. 7H). Furthermore, this difference was maintained in MEFs co-treated with hexadecenal and ABT‑737 (Fig. 7H, I). Taken together, these results suggest that BAXP168G is insensitive to hexadecenal and synergy with BIM due to dysfunction of the BAF. Furthermore, we posit that the increased flexibility in the α8−9 loop (by substituting a rigid proline for glycine) may also alter the mobilization of α8 and subsequent BAF exposure following BIM triggering in addition to deforming the BAF structure through allosteric side chain reorganization.

Methods
Experimental models
Bacterial cell lines. For recombinant protein generation, One ShotTM BL21 (DE3) Chemically Competent E. coli were purchased from Invitrogen/Thermo Fisher Scientific (Cat. No. C600003). Cells were grown in BDD ifco TM Terrific Broth (Cat. No. BD243820, Thermo Fisher Scientific) media supplemented with 1% glycerol at 37°C with shaking at 220 rpm.

Cell lines
Bax+/+Bak+/+ and Bax−/−Bak−/− SV40-transformed MEFs were obtained from ATCC (Manassas, VA, USA); Bim−/−Bid−/− SV40-transformed MEFs were provided by Dr. Douglas Green (St. Jude Children’s Research Hospital); Bax−/−Bak−/− double knockout MEFs reconstituted to express wild type or P168G BAX were provided by Dr. Evripidis Gavathiotis (Albert Einstein College of Medicine) and BAX expression was confirmed by western blot and GFP positivity from the pBabe IRES‑GFP vector backbone. Cells were cultured in high-glucose DMEM (Cat. No. 10-017-CV, Corning, Manassas, VA, USA) containing 10% FBS (Cat. No. 10438-026, Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine (Cat. No. 25030-081, Life Technologies, Carlsbad, CA, USA), and 1× Penicillin/Streptomycin (Cat. No. 10378-016, Life Technologies, Carlsbad, CA, USA), and grown in humidified incubators at 37°C with 5% CO2. All cell cultures were maintained in mycoplasm-free conditions as verified by the HEK-Blue Detection Kit (Cat. No. hb-det2, Invivogen, San Diego, CA, USA).

Recombinant protein and peptides
Expression vectors and site-directed mutagenesis. Recombinant human BAX was expressed and purified using an intein-chitin binding domain (CBD) tag from a pTYB1 vector (BAX cDNA inserted into Nde/SapI cloning site, with a C-terminal tag)44. BAX mutants (e.g., C62S and C126S double mutant, BAX2S; L59C, C62S, C126S, and L162C quadruple mutant, BAXα8) were generated by site-directed mutagenesis of the WT construct. Amplification of the de novo plasmids was accomplished using CloneAmp HiFi PCR Premix (Cat. No. 639298, Takara Bio, Mountain View, CA, USA) and thermo-cycled as follows: 1× [98°C for 2 min]; 30× [98°C for 10 s, 55°C for 30 s, 72°C for 8 min]; and 1× [72°C for 10 min]. Parental plasmids were digested by 1 μl DpnI enzyme (Cat. No. R0176, New England Biolabs, Ipswich, MA, USA) at 37°C for 15 min, followed by purification using the QIAquick PCR Purification Kit (Cat. No. 28104, Qiagen, Hilden, Germany). Sequences were verified by Genewiz Sanger sequencing using the Universal T7 primer (5’-TAATACGACTCACTATAGGG-3’).

Recombinant BAX expression and purification. One Shot™ BL21 (DE3) chemically competent E. coli (Cat. No. C601003, Thermo Fisher Scientific) were transformed with the pTYB1-BAX construct in LB broth and grown on agar plates supplemented with 100 μg/mL carbenicillin at 37°C. Starter cultures were grown in Terrific Broth (TB) supplemented with 0.4% glycerol and 100 μg/mL carbenicillin at 30°C for 14–16 h to an optical density at 600 nM (OD600) of approximately 1.5; then diluted with 4× volume of TB broth and expanded at 37°C for 3–4 h until the culture achieves a target OD600 of 2–3. Recombinant BAX expression was induced with 1 mM IPTG at 30°C for 6 h. Bacteria were pelleted, resuspended in lysis buffer (50 mM K2PO4, 50 mM NaH2PO4, 500 mM NaCl, 1 mM EDTA, 0.1 mM AEBSF) supplemented with a Pierce protease inhibitor tablet (Cat. No. A32965, Thermo Fisher Scientific), and lysed with a probe sonicator (e.g., Dismembrator 505, Thermo Fisher Scientific) for 20 min. Lysates were centrifuged at 42,000×g at 4°C for 1 h to pellet debris and recombinant intein-CBD-tagged BAX was captured from the supernatant by chitin affinity chromatography at 4°C according to the manufacturer’s instructions (Cat. No. S5551, New England Biolabs, Ipswich, MA, USA). On-column intein cleavage of the CBD tag was achieved by incubation with DTT (50 mM) at 4°C for at least 16 h, followed by elution in Gel Filtration Buffer (20 mM HEPES [pH 7.4], 150 mM NaCl). Recombinant BAX protein (now without a tag or exogenous residues) was further purified by size-exclusion chromatography using Gel Filtration Buffer on a HiLoad 16/600 or 26/600 Superdex 200pg column (Cat. No. 28989335, Cytiva) using an ÄKTA pure™ 25 L1 (Cat. No. 29018225, Cytiva) at 4°C according to the manufacturer’s instructions. Fractions containing BAX protein were combined and concentrated using Amicon Ultra-4 centrifugal filter (Cat. No. UFC801024, Millipore Sigma) to a stock concentration of ~30 μM, prior to snap freezing with liquid nitrogen, and storage at −80°C. A detailed version of this protocol has been published65.

Oxidizing disulfide tether BAX mutants. For the BAXα8 mutant, the nascent protein was purified as the reduced form due to incubation with DTT during on-column intein cleavage; to generate a disulfide tether (“locked BAXα8”), BAX protein was oxidized in 1 mM Dichloro(1,10-Phenanthroline)Copper(II) (Cat. No. 362204, Millipore Sigma-Aldrich) at 4°C for 15 min, purified by Superdex 75 Increase 10/300 GL column (Cat. No. 29148721, Cytiva), and only monomeric species were pooled and concentrated to make protein stocks.

Other reagents. Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl‑xLΔC, aa 2–212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Lipid aldehyde preparation
2-trans-hexadecenal (16:1 aldehyde) was purchased in powder form (Cat. No. 857459P, Avanti Research) and reconstituted in pre-warmed (37°C) anhydrous DMSO (Cat. No. D12345, Thermo Fisher Scientific) to a concentration of 50 mM using a Hamilton syringe. The reconstituted 2t‑hexadecenal was aliquoted into single-use glass aliquots and stored at −80°C. To avoid precipitation/coagulation of 2t‑hexadecenal, stocks were serially diluted first in pre-warmed DMSO, then diluted into pre-warmed aqueous assay buffer to use as a stock for the experiment. This was typically accomplished as follows: 2.5 μl of 50 mM 2t‑hexadecenal stocks were two-fold diluted four times in DMSO (to 40 μl of 3.13 mM), then diluted twice in the selected assay buffer (first with 40 μl, then with 45 μl) to achieve a working stock of 125 μl of 1 mM 2t‑hexadecenal in 32% DMSO/buffer. Working stocks of 1 mM 2t‑hexadecenal were placed in a water bath sonicator for 15 min to aid in solubilization and then further diluted to the desired concentration and acceptable DMSO background for experiments. For cell death experiments, 2t‑hexadecenal was prepared in 0% FBS media to prevent binding to serum proteins (see SPARKL method below). For aldehyde modification, (2‑hydroxyethyl)hydrazine (Cat. No. 043793.09, Thermo Fisher Scientific) was added to stock 2t‑hexadecenal at a ratio of 30−50 molar excess and reacted at 37°C for 1 h. The mass of the resultant hexadecenal-hydrazino product (2t16‑NN‑EtOH) was confirmed by mass spectrometry (see below) and used to validate the reagent for BAX function assays.

Hexadecanal (16:0 aldehyde) was purchased as 1 mg/ml (4.16 mM) in methylene chloride solution (Cat. No. 857458M, Avanti Research), aliquoted into 25 μl, and stored at −80°C. Prior to use, hexadecanal was first diluted with pre-warmed DMSO to a concentration of 3 mM, then diluted to 1 mM with pre-warmed assay buffer of choice, sonicated for 15 min, and then further diluted for experiments. The remaining alkenals were purchased in liquid form (see Table 1), and their molar concentrations were calculated based on their density, purity, and molecular weight. Short-chain alkenals (2t5–2t10) were diluted with DMSO to a stock concentration of 1 M, aliquoted, and stored at −80°C. Prior to use, stocks were further diluted with DMSO to 320 mM and then in assay buffer to a final concentration of 1 mM with 32% DMSO (v/v). Long-chain alkenals (2t11–2t13) were more likely to coagulate and thus were prepared following the exact same procedure as the 2t‑hexadecenal preparation.

Real-time live-cell detection of apoptosis (SPARKL)
Cell culture and treatments. MEFs were seeded at 2–4×103 cells per well in 96-well tissue culture treated plates and left to adhere for 18–24 h. Prior to live-cell imaging, growth media was replaced with phenol red-free DMEM as follows: first, 50 μl (50% total volume) of 0% FBS DMEM containing 2t‑hexadecenal treatments was added to wells for 15−30 min to allow uptake; next, 50 μl of 10% FBS DMEM containing additional treatments and fluorescently-labelled Annexin V (50 nM or ~1.8 ng/μl) was added to wells to constitute serum levels. Recombinant Annexin V was generated in-house as previously described23,66,67. Immediately following treatments, plates were subjected to real-time fluorescence microscopy, automated data collection, and analysis.

Data acquisition and event detection with an IncuCyte ZOOM. SPARKL experiments performed using an IncuCyte ZOOM (Model 4459, Sartorius, Göttingen, Germany) were housed in a humidified tissue culture incubator maintained at 37°C with 5% CO2. Images were collected every 2 h for 24 h using a 10× objective, and a single field of view was collected per well. Brightfield and green channels (Ex: 440/80 nm; Em: 504/44 nm; acquisition time: 400 ms) were collected at 1392×1040 pixels at 1.22 μm/pixel. Automated event detection was accomplished using the ZOOM software (v2018A) and user-defined processing definition using images collected using the relevant cell lines and fluorescent reporters. Processing definition settings were as follows: Parameter, Top-Hat; Radius (μm), 25; Threshold (RFU), 3; Edge Sensitivity, −30; Area (μm2), >100. Kinetic data are graphed as the calculated events per well metric provided by the ZOOM software. The y-axis scale was determined for each experiment using parallel internal control treatments to assess maximal apoptotic death. Data are the mean of replicates and are representative of at least three repeated and reproduced assays. A detailed explanation of this method23 and protocol68 have been published.

Data acquisition and event detection with a Cytation 7. SPARKL experiments performed using a Cytation 7 equipped with an inverted imager (Model CYT7UW-SN, BioTek/Agilent, Santa Clara, CA, USA) tethered to a BioSpa 8 automated incubator (Model BIOSPAG-SN, BioTek/Agilent, Santa Clara, CA, USA), where cells were maintained in a humidified environment at 37°C with 5% CO2. Bright field and red channel images were collected every 2 h for at least 24 h using a 10× objective with a 75% wide field of view crop (1045×1045 μm), and a single field of view was collected per well using a laser autofocus (Part No. 1225010). Bright field images were acquired with the following settings: LED intensity, 7; Integration Time, 100 msec; Camera Gain, 19. Red channel images (Ex: 586/18 nm; Em: 647/57, Part No. 1225102) were acquired with the following settings: LED intensity, 8; Integration Time, 20 msec; Camera Gain, 19. Automated red event detection was accomplished using the Gen5 software (v3.12) and a cellular analysis data reduction profile with the following settings: Threshold, 5000 RIU; Background, Dark; Split Touching Objects, yes; Fill holes, yes; Background Flattening Size, auto (270 μm); Image Smoothing Strength, 0; Background Percentage, 5%; Minimum Object Size: 5 μm; Maximum Object Size: 90 μm. Kinetic data are graphed as events per image. The y-axis scale was determined for each experiment using parallel internal control treatments to assess maximal apoptotic death. Data are the mean of replicates and are representative of at least three repeated and reproduced assays.

Large unilamellar vesicle (LUV) permeabilization assays
LUV composition and preparation. LUVs and permeabilization assays were prepared as similarly described9,69. Briefly, chicken egg phosphatidylcholine (Cat. No. 840051C, Avanti Research), chicken egg phosphatidylethanoloamine (Cat. No. 840021C, Avanti Research), porcine brain phosphatidylserine (Cat. No. 840032C, Avanti Research), bovine liver phosphatidylinositol (Cat. No. 840042C, Avanti Research), and cardiolipin (18:1) (Cat. No. 710335C, Avanti Research) were combined at a ratio of 48:28:10:10:4 (5 mg total), dried under N2 gas, and resuspended in LUV buffer (pH 7.2, 200 mM KCl, 5 mM HEPES, 0.2 mM EDTA) containing a polyanionic dye (12.5 mM ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (45 mM DPX: p-xylene-bis-pyridinium bromide) using a water bath sonicator. Unilamellar vesicles were formed by 31 extrusions of the suspension through 1.0 μm polycarbonate membranes (Cat. No. 610010, Avanti Research, Alabaster, AL, USA). The unincorporated DPX and ANTS were removed by using a 10 ml Sepharose S-500 gravity flow column. LUVs were used within 2 weeks of preparation to avoid significant liposome degradation.

LUV assays. Working stocks of recombinant proteins, peptides, and lipids were prepared in LUV buffer at 4× their intended final concentrations. LUVs from the preparation stock (20×) were diluted five-fold in LUV buffer to generate a working stock (4×) for assays (equivalent to each sample receiving 5 μl undiluted LUVs). In an opaque black polystyrene 96‑well plate (Cat. No. 3915, Corning), lipids or peptide titrations were generated via in-well serial dilutions and LUV buffer was added to any control wells requiring volume compensation. Samples were assayed in 100 μl total volume with 25 μl of each component sequentially added to achieve desired 1× concentrations. A typical assay was prepared as follows: 25 μl of titrant is prepared in the well; then 25 μl of peptide, reagent, or buffer is added; then 25 μl of recombinant BAX; and finally, 25 μl of diluted LUVs. Plates were immediately subjected to fluorescence analyses for 90 min using either a Synergy H1 or Cytation 5 multi-mode microplate reader (Agilent/BioTek, Santa Clara, CA, USA) using the following parameters: Read interval, 55 sec; Protocol, [37°C, 2 sec linear shake, read]; Excitation, 355/20 nm; Excitation, 520/20 nm; Gain (voltage), 100; Optics position, top; Read height, 5.5 mm. Kinetic data represents the mean of triplicate samples and are representative of at least three repeated and reproduced assays using separate protein aliquots and LUV preparations. Every assay included a 1% CHAPS positive control to determine maximum signal. Normalized endpoint data (% permeabilization) were calculated in Prism (Graphpad) using the minimum value of the buffer control (as 0%) and the mean value of LUVs solubilized in 1% CHAPS (as 100%) measured during the entire assay (Equation 1). Appropriate concentrations of BAX were determined by extensive protein titrations for each preparation of recombinant protein and LUVs; typically, the highest BAX concentration exhibiting minimal permeabilization was selected for subsequent assays to ensure adequate signal and dynamic range for activation studies. Data are the mean of replicates and are representative of at least three repeated and reproduced assays.

LUV data analysis. Kinetic LUV permeabilization was fit with Prism (Graphpad) nonlinear regression analysis using a modified 5-parameter sigmoidal model (Equation 2), with S≤10 and T≥B. The formula for maximal rate, or slope at the inflection point (IP), was derived from the second derivative of the function and incorporated into the analysis report as an exported metric (Equation 3). Bliss synergy scores were determined for normalized endpoint LUV permeabilization data using the SynergyFinder Plus web application (www.synergyfinder.org) and graphed in Prism70.

Microscale thermophoresis (MST)
MST experiments were carried out on a NT.115 (NanoTemper GmbH, Munich, Germany) instrument and performed in MST buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20). EC50 values were calculated using the NanoTemper software. The recombinant human BAX protein was labeled with Alexa Fluor-647 NHS ester by incubating 60 μM of the fluorescent dye with a 20 μM of BAX protein for 30 min in the dark. The labeling mixture was subsequently applied to a G-25 gravity flow column (GE Healthcare) that had been equilibrated with MST buffer. After elution from the G-25 column, the concentration of the labeled protein was quantified spectrophotometrically, snap frozen in liquid nitrogen, and stored as single-use aliquots at −80°C. Labeled BAX protein was treated with 0.002% CHAPS to prevent activation-induced BAX aggregation upon ligand binding. Curve fitting was conducted using Prism (Graphpad) and only applied to X values from 5−35 s using Equation 4, and the K constant was calculated using Equation 5. Data are representative of at least three repeated and reproduced assays.

Liquid chromatography-mass spectrometry (LC-MS)
Sample preparation and handling. Working stocks of recombinant BAX protein and 2t‑hexadecenal were prepared in Gel Filtration Buffer. 30 μl of 5 μM recombinant BAX (~3 μg) was treated as indicated in duplicate and incubated at 37°C for 2 h. Samples were then incubated in 8 M urea buffer (with 50 mM NH4HCO3) at 57°C for 1 h, and alkylated in the dark with 30 mM iodoacetamide for 30 min at 25°C. Samples were then digested using 1:100 (w/w) trypsin at 37°C for 4 h, with an additional round of 1:100 trypsin at 37°C for 16–18 h. After proteolysis, the peptide mixtures were desalted by self-packed stage-tips or Sep-Pak C18 columns (Waters) and analyzed with a Vanquish Neo UHPLC System that is coupled online with a Orbitrap Exploris 480 mass spectrometer (Thermo Fisher). Briefly, desalted Nb peptides were loaded onto an analytical column (C18, 1.7 μm particle size, 150 μm × 5 cm; Ion Opticks) and eluted using a 20 min liquid chromatography gradient (3–10% B, 0–2 min; 10–40% B, 2–12 min; 40–80% B, 12–14 min; 3% B, 14–20 min). Mobile phase A consisted of 0.1% formic acid (FA), and mobile phase B consisted of 0.1% FA in 80% acetonitrile (ACN). The flow rate was 1 μl/minute. The Orbitrap Exploris 480 instrument was operated in the data-dependent mode, where the top 20 most abundant ions (mass range 200–1800, charge state 2–6) were fragmented by high-energy collisional dissociation (HCD). The target resolution was 120,000 for MS and 15,000 for tandem MS (MS/MS) analyses. The maximum injection time for MS/MS was set at 100 ms. Data were replicated using different protein batches.

Proteomic data analysis. Raw data collected by LC-MS was searched against the Uniprot reviewed Human protein sequences database retrieved on 01 March 2024 with decoys and common contaminants appended using FragPipe (v22.0). A labile search was performed in MSFragger71 without diagnostic and Y ions specified. Mass offsets were set restricted to cysteines. The offset list was set at 0 (no modification) and 238.22967 (monoisotopic mass of 2t‑hexadecenal) and replacing the fixed cysteine carbamidomethylation with a variable one. “Write calibrated MGF” was turned on for the PTM-Shepherd72 diagnostic feature mining module, and “Diagnostic Feature Discovery” in PTM-Shepherd was enabled with default parameters. Peptide and protein levels were performed using label-free quantification (LFQ) algorithms in IonQuant73. After MS search with MSFragger, raw files and identification files were imported into PDV74 (https://github.com/wenbostar/PDV) for MS spectra annotation.

Time of flight (TOF) mass spectrometry
Small molecule mass spectrometry. Small molecules were analyzed by using an Agilent 1200 series system with DAD detector and a 2.1×50 mm InfinityLab Poroshell 120 EC‑C18 column for chromatography. Samples (0.5 μl) were injected onto a C18 column at room temperature with the flow rate of 0.6 ml/min. Chromatography was performed with the solvent as follows: water containing 0.1% formic acid and 3% acetonitrile was designated as Solvent A while acetonitrile containing 0.1% formic acid was designated as solvent B. The linear gradient was set such that 5% B was used from 0–0.5 min, 5–100% B from 0.5–2.8 min, 100% B from 2.8–4.2 min, 100–5% B from 4.2–4.3 min, and 5% B from 4.3–5 min. High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent 6230 TOF with an electrospray ionization (ESI) source. Results were replicated in three repeated experiments.

Native intact protein mass spectrometry. Working stocks of recombinant BAX protein (10−20 μM) were treated with DMSO vehicle, 2t‑hexadecenal (10−200 μM), or iodoacetamide (50 mM) were prepared in 20 μl of 10 mM HEPES buffer and reacted for 1 h. Intact MS was performed by an Agilent 12300 series system with DAD detector and a 2.1×150 mm C18 column for chromatography. Samples (10 μl) were injected onto a C18 column at room temperature with the flow rate of 0.4 ml/minutes. Chromatography was performed with the solvent as follows: water containing 0.1% formic acid and 3% acetonitrile was designated as Solvent A while acetonitrile containing 0.1% formic acid was designated as solvent B. The linear gradient was set such that 5% B was used from 0–1 min, 5–70% B from 1–6 min, 70% B from 6–8 min, and 5% B from 8–12 min. Mass spectra data was acquired in positive ion mode using an Agilent 6230 TOF with an electrospray ionization (ESI) source. MS spectra (m/z 600−1600) were deconvoluted by Agilent MassHunter BioConfirm v12.0 using a mass range of 16000−25000 daltons. Results were replicated in four repeated experiments.

Thermal shift differential scanning fluorimetry
Working stock of recombinant BAX proteins, SYPRO Orange dye (Cat. No. S6650, Thermo Fisher Scientific), and 2t‑hexadecenal were prepared in gel filtration buffer. The assay was performed in 100 μl 96-well PCR plates using a total assay volume of 50 μl. Twenty microliter of 2.5× 2t‑hexadecenal titrations were generated via in-well serial dilutions and buffer was added to any control wells requiring volume compensation. 20 μl of 2.5 μM BAX and 10 μl of 20× SYPRO Orange were sequentially added to the wells, thoroughly mixed by pipetting, and the plate was centrifuged to recollect sample in the bottom of the well and remove any trapped bubbles. The PCR plate was then sealed with optically clear adhesive sheet and subjected to fluorescence spectroscopy using an Applied Biosystems ViiA7 real-time PCR instrument (Thermo Fisher Scientific). Temperature started at 25°C and increased to 95°C at a rate of 1% per minute. Data was collected as normalized fluorescence at each step of the thermal ramp and the melting temperature was determined as the maximum first derivative value. Data shown are the average of at least three replicates with error bars denote the SEM, and representative of at least three repeated and reproduced assays.

Fluorescence polarization ligand assay to monitor BAX early-activation (FLAMBE)
Assay setup. Working stocks of recombinant BAX protein, peptides, and lipids were prepared in 0.5× PBS buffer at 4× their intended final concentrations. 5-TAMRA labeled BAK-BH3 peptide (Cat. No. AS-64590, AnaSpec) was diluted to a 200 nM working stock (4×). In an opaque black polystyrene 96‑well plate (Cat No. 3915, Corning), lipids or peptide titrations were generated via in-well serial dilutions and buffer was added to any control wells requiring volume compensation. Samples were assayed in 100 μl total volume with 25 μl of each component sequentially added to achieve desired 1× concentrations. A typical assay was prepared as follows: 25 μl of titrant is prepared in the well; then 25 μl of peptide, reagent, or buffer is added; then 25 μl of recombinant BAX; and finally, 25 μl of BAKTAMRA. Plates were immediately subjected to spectrometry analyses for 60 min using a Synergy H1 multi-mode microplate reader equipped with a red polarization filter (8040562, Agilent/BioTek, Santa Clara, CA, USA) using the following parameters: Read interval, 30 s; Protocol, [25°C, 2 s double orbital shake, read]; Excitation, 530/25 nm; Excitation, 590/35 nm; Mirror, 570 nm; Gain (voltage), 50; Optics position, top; Read height, 7.0 mm. Polarization (expressed as milli-Polarization (mP) units) is derived from measured parallel (||) and perpendicular (⟂) emission intensities (I) and was calculated by the Gen5 software (Agilent/BioTek); Polarization can also be calculated manually using Equation 6. Kinetic data represents the mean of triplicate samples and are representative of at least three repeated and reproduced assays using separate protein aliquots. Appropriate concentrations of BAX and peptides were determined by extensive protein titrations for each preparation of recombinant protein; typically, the highest BAX concentration exhibiting no BAKTAMRA dissociation was selected for subsequent assays to ensure adequate signal and dynamic range for activation studies. Similarly, concentrations of direct activators were selected from extensive titration experiments to determine the appropriate concentration for each experimental setup. Data are the mean of replicates and are representative of at least three repeated and reproduced assays.

Kinetic FP data parameterization. Parameterized FLAMBE data was derived from the average of replicates to reduce data noise. For each condition, Tmax was identified as the timepoint with the highest Polarization and EP was the endpoint Polarization value recorded. Tmax of the BAKTAMRA control or any sample exhibiting no binding kinetics during the assay was set to 0 to avoid misidentification due to noise. Each parameterized metric was normalized to metrics from the BAKTAMRA and BAX control conditions, as 0 and 1, respectively. The shift magnitude of parameterized FLAMBE data was calculated using the distance between conditions without and with BIM-BH3 treatment (Equation 7). Area under the curve (AUC) was calculated using Prism (Graphpad) and normalized to the controls. A detailed explanation of this method8 and protocol75 have been published.

Competition FP assay. BCL‑xL competitive binding FP assays were conducted similarly to FLAMBE assays with some modifications. BCL‑xL, 2t‑hexadecenal, and BAKTAMRA were prepared as 4× working stocks in a modified FP assay buffer (20 mM sodium phosphate buffer [pH 7.4], 50 mM NaCl, 1 mM EDTA, 0.05% pluronic F-68)76. 2t‑hexadecenal titrations were prepared in-well and diluted with 25 μl buffer; separately, recombinant BCL‑xL and BAKTAMRA stocks are combined at a 1:1 volume ratio (resulting in a 2× stock) and 50 μl is added to sample wells immediately followed by spectrometry.

Fluorescent size-exclusion chromatography
Alexa Fluor™ 488 NHS ester (AF488)-labeled BAX was generated according to the manufacturer’s instructions (Thermo Fisher Scientific, Cat. No. A20000) at a 20:1 dye:protein molar excess. Excess dye was removed by gel filtration with a Superdex 75 Increase 10/300 GL column (Cat. No. 29148721, Cytiva) equilibrated with Gel Filtration Buffer using an ÄKTA pure™ 25 L1 (Cat. No. 29018224, Cytiva) at 4°C according to the manufacturer’s instructions. 0.5 ml fractions were collected and fractions containing monomeric AF488-labelled BAX were combined, quantified, aliquoted, and stored at −80°C. For experiments, 600 μl of 400 nM 488-labelled BAX was treated as indicated at 25°C for 1 h and protected from light. Following treatment, BAX was subjected to size exclusion chromatography using the above parameters. For detection, 0.2 ml of eluate from the indicated fractions was transferred to a black 96 well-plate and analyzed for fluorescence by a Cytation 5 multi-mode plate reader (BioTek/Agilent, Santa Clara, CA, USA) using the following parameters: Excitation, 495/10 nm; Emission, 520/10 nm; Gain (volage), 100. Results were replicated in at least three repeated experiments.

2D HSQC NMR spectroscopy
15N-labeled BAX protein was prepared at 40 μM in 15 mM sodium phosphate (pH 7.0), 50 mM NaCl. Samples were prepared and immediately subjected to spectroscopy. Data were acquired on Bruker 600-MHz spectrometer equipped with a cryoprobe. Two-dimensional 1H-15N correlation spectra were acquired at 25°C using standard Bruker pulse sequences using 128 scans, 2,048 × 200 complex points and spectral windows of 14×35 p.p.m. in the 1H and 15N dimensions, respectively. Samples did not exhibit any treatment-specific loss of signal or peak intensities suggestive of protein loss (due to either degradation or activation-induced multimerization). Spectra were processed using TOPSpin (Bruker Biospin, MA, USA) and analyzed with CARA software (cara.nmr.ch)77. Peaks were assigned using assignments from a previous publication44. The weighted average chemical shift perturbation (CSP) difference (Δδ) was calculated using Eq. 8. The absence of a value indicates no CSP difference, the presence of a proline, or a resonance that is overlapped or missing and therefore could not be confidently assigned and not used in the analysis. The significance threshold for CSPs was calculated based on the average chemical shift across all measurable residues plus 1 or 2 standard deviations, in accordance with standard methods. Mapping of CSP data onto the BAX structure (PDB: 1F16) was performed with PyMOL (Schrödinger, LLC).

In silico docking and molecular modeling
Unconstrained global rigid-body docking. The molecular interactions between the alkenals and BAX were modeled using the EADock DSS version of the SwissDock web server (www.swissdock.ch)78,79. The structure PDB files and the alkenal mol2 files were obtained from the Protein Data Bank and the ZINC database, respectively (see Table 2). Output files were split into individual clusters and visualized using PyMOL (Schrödinger, LLC). Quantification of alkenal binding was accomplished by manual inspection of each cluster group and the total number of binding modality outputs for each job. Cavity determination and visualization was performed with PyMOL using a cavity radius of 3 and a cavity cutoff of −5.5; additional spaces were excluded to enhance visualization.

Virtual mutagenesis and guided molecular docking. Virtual mutagenesis of BAX structures was performed using the mutagenesis wizard in PyMOL (v3.1, Schrödinger, LLC) and selecting rotamers that did not conflict with surrounding side chains; the resulting structures were utilized in downstream investigations. NMR-guided molecular docking of 2t‑hexadecenal on BAX was performed using Glide (v2023‑1 build 128, Schrödinger, LLC) with extra precision (XP) and a receptor grid generated in the center of the BAF (13×13×13 Å inner box, 10 Å outer box), with no additional constraints. Wild type and mutant structures had α8 residues (157−163) removed and were prepared using the Protein Prep Wizard, assigning partial charges with EPIK (Schrödinger, LLC), and aligned against the 1F16 structure to ensure consistency of BAF coordinates for grid generation. The 2t‑hexadecenal structure was converted to 3D and prepared for docking using LIGPREP (v2023‑1 build 128, Schrödinger, LLC); the structure for the 2t‑hexadecenal-hydrazino-product (2t16‑NN‑EtOH) was drawn in MAESTRO and similarly prepared. The lowest energy docking pose is visualized, was representative of all output poses, and various scoring metrics are reported. Interaction fingerprints were generated for residues exhibiting overall interaction with any of the output poses. Interaction comparisons were calculated as the fraction of total poses interacting with a residue, relative to the WT:2t‑hexadecenal interaction, and color-coded for visualization in PyMOL (Schrödinger, LLC). Interaction diagrams were generated and exported from MAESTRO (Schrödinger, LLC) and then redrawn in Inkscape (www.inkscape.org) to aid in visualization.

SDS-PAGE and western blotting
Bax−/−Bak−/− MEFs, or MEFs reconstituted to express WT or P168G BAX, were harvested with 0.25% Trypsin-EDTA and pelleted at 800×g for 5 min. Cells were lysed with 1× RIPA lysis buffer supplemented with protease inhibitors (HALT Tablet, Cat. No. 87786) and phosphatase inhibitors (Cat. No. k1015b, APExBIO) on ice for 20 min, pelleted at 21,000×g for 10 min at 4°C, and supernatant was collected for protein quantification by BCA assay (Cat. No. 23225, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Sample concentrations were equilibrated in lysis buffer, combined with 4× Laemmli sample loading buffer supplemented with DTT, subjected to SDS-PAGE in a 12.5% polyacrylamide gel, and transferred to nitrocellulose by standard western blot conditions. Membranes were blocked in 5% milk in 1× TBS buffer supplemented with 0.1% Tween-20, incubated with primary antibodies (1:500-1000 in blocking buffer; incubated for 16−18 h at 4°C) and secondary antibodies (1:4000 in blocking buffer; incubated for 1 h at 25°C), followed by standard enhanced chemiluminescence detection (Cat. No. WBLUF0100, Sigma-Aldrich, St. Louis, MO, USA). Antibodies: BAX, 1:500 dilution (Clone 2D2, Cat. No. sc-20067, Santa Cruz Biotechnology, Dallas, TX, USA); GAPDH, 1:1000 dilution (Clone 1E6D9, Cat. No. 60004, Proteintech, Rosemont, IL, USA); m-IgGk BP-HRP secondary antibody (Cat. No. sc-516102, Santa Cruz Biotechnology, Dallas, TX, USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the authors aim to characterize the mechanism of activation of the BAX protein by 2t-hexadecenal. The authors discovered a binding pocket, which they termed the \"BAX actuating funnel\", where they propose lipid molecules binds, which leads to conformational change, a key step in BAX activation.\nThe authors used several types of biophysical technique to characterize BAX-2t-hexadecenal interaction, such a microscale thermophoresis (MST), nuclear magnetic resonance spectroscopy, mass spectrometry, and fluorescence polarization method (referred here as FLAMBE). The authors also made and purified several mutants of BAX and present some mammalian cell culture work.\nOverall, the paper address one key question: how is BAX activated, and how is it becoming anchored to the membrane in the mitochondria. Previous studies have shown that a cysteine present in BAX is being covalently modified by the lipid, which lead to conformational change and membrane anchoring, and eventual MOMP in the mitochondria. This is paper challenges this concept and propose an alternative mechanism of BAX activation anchorage to the membrane, through non-covalent interaction of lipids in the “BAF” hydrophobic pocket.\nThe article is well written, the experiments sound, and the figures are very nicely presented. The authors also present a good discussion presenting their results in the context of previously contradictory results. Finally, while this work is perhaps a little controversial, I believe it is worth publishing and propose an alternative avenue of BAX activation.\nComments:\n• Fig. 1B. Why is the control DMSO (for 40 uM) in the SPARKL analysis show such strong signal, more than the ABT 20 uM? This seems concerning.\n• Line 111. “[we] observed a loss of apoptosis at lower concentrations of ectopic hexadecenal co-treated with ABT-737, suggesting that BAX activation was part of the underlying mechanism (Figure 1B). Finally, we replicated this experiment in Bax−/−Bak−/− DKO MEFs and observed no cell death (Figure 1C). Collectively, these data support the conclusion that hexadecenal induces apoptotic cell death by acting on effector BCL‑2 family proteins.”\n--> While there is nothing wrong with these results, I am puzzled at the conclusion. My conclusion is that ABT-737 obviously doesn’t kill Bax−/−Bak−/− DKO.\n• Line 131, Fig. 1F. “Furthermore, the synergy with hexadecenal was observed with mildly activating concentrations of BIM‑BH3 as well (Figures 1F, S1C).”\n--> Could is simply be additive? In order to claim synergy, I think the authors need to present the synergistic calculation…\n• Line 152. “We detected peptide fragments covering both cysteines (C62, C126) and observed no alkylation by hexadecenal (m+238.229 Da);”\n--> Is it possible trypsin can’t cleave after the lysine at close proximity to the modification?\n• The authors report chemical shift changes – but what about comparing line broadening between the mutants? At the residue level, or simply using 1D HN relaxation envelop? There seem to be a loss in chemical shift dispersion (Supp. Fig. 4A)\n• Line 330” I don’t like the term “unbiased docking assessments”. Nothing is unbiased.\n• Line 399. Typo. “Directly comparison”\n• Line 475. “We also observed that short-chain alkenals did not activate BAX, which aligns with a prior study that also demonstrated increased reactivity with short-chain alkenals.”\n--> Increased reactivity doesn’t lead to increase BAX activation?", "reviewer_2": "The study by Gelles et al. presents a multidisciplinary investigation into how the lipid metabolite 2t-hexadecenal enhances BAX activation, a central event in mitochondrial apoptosis. The authors combine NMR, computational modeling, biochemical and cellular assays, and mutagenesis to provide mechanistic insight into this interaction. They show that 2t-hexadecenal binds a novel binding region on BAX, a hydrophobic cavity formed by helices α5, α6, and gated by α8, which they term the BAX Actuating Funnel (BAF). Their data suggest that 2t-hexadecenal binds non-covalently at this site, priming BAX for activation, particularly in the presence of the BH3-only activator BIM. Further, they show that α8 helix mobility and alkenal chain length are critical determinants of lipid-mediated BAX activation. The study is thorough and well-controlled, offering a rational model for how 2t-hexadecenal lipid metabolite can modulate BAX conformational dynamics to facilitate BH3-mediated BAX activation leading to mitochondrial outer membrane permeabilization (MOMP).\nOverall, the manuscript offers a novel conceptual model for liped-mediated BAX activation and a strong case for the biological relevance of the newly identified BAF site, that may be exploited for therapeutic modulation using small molecules. However, I have listed several issues below that should be addressed to strengthen the manuscript and validate the conclusions before publication.\n1. The authors report a dissociation constant (Kd) of 0.182 μM for 2t-hexadecenal binding to BAX (MST assay), yet concentrations >> 6.5–10 μM are used in FLAMBE, liposomal, and cellular assays, including in the presence of BIM BH3. The rationale for this discrepancy should be clearly explained. Have the authors used concentrations near the Kd concentration? Why functional effects require supersaturating conditions?\n2. The authors report a non-covalent interaction between 2t-hexadecenal and BAX. However, prior studies from two independent groups have provided convincing evidence for covalent modification of BAX by 2t-hexadecenal. Did the authors use comparable experimental conditions and mass spectrometry techniques to exclude the possibility of covalent binding in their assays?\n3. Given the known reactivity of alkenal metabolites, can the authors confirm the purity and structural integrity of the 2t-hexadecenal used in their experiments? Can they provide batch consistency and analytical validation (e.g., NMR, MS) to help rule out variability of the 2t-hexadecenal as a factor in observed differences compared to previous studies.\n4. The manuscript briefly suggests that NP-40 (a non-ionic detergent) may explain some discrepancies with earlier findings. The authors should elaborate on this point. How might NP-40 influence lipid-protein interactions in lysates or buffers in a way that affects covalent modification?\n5. The authors note that the BAF cavity is present in previously reported structures of BAX bound to BID BH3 or BAX BH3 domains and the domain-swapped dimers of BAX. The authors should include a comparative structural figure highlighting the BAF site across these BAX structures for additional clarity and support of this conclusion.\n6. Figures lack detailed information about the number of replicates and statistical analyses performed. The authors should include this information to figure legends wherever applicable.", "reviewer_3": "Gelles et al. investigate the mechanism by which the bioactive sphingolipid metabolite trans-2-hexadecenal (2t-16) promotes BAX activation, a process relevant to mitochondrial outer membrane permeabilisation and apoptotic cell death. Employing biochemical, structural, and computational approaches, the authors identify a hydrophobic region of BAX as a putative binding site for non-covalent interaction with 2t-16. The authors propose that this interaction, particularly in the presence of the BH3-only activator BIM, potentiates early BAX conformational changes and promotes membrane permeabilisation, without invoking the covalent mechanism elucidated in prior studies.\nWhilst the study employs several creative indirect methods and identifies a potential lipid interaction site, the central claim that 2t-16 operates principally through a non-covalent mechanism rests on incomplete and, at times, internally inconsistent evidence. Given the well-established reactivity of α,β-unsaturated aldehydes and the membrane-embedded nature of lipid regulation, much of the data—derived largely from high-concentration solution-phase lipid treatments and rigid-body docking to an inactive BAX structure—appears inconsistent with the proposed model. The findings may ultimately align better with a dual mechanism involving both covalent and non-covalent features, as is commonly observed in physiological and pharmacological covalent protein regulation.\nMoreover, the study is marked by a considerable degree of speculation, with data interpreted selectively to support the authors’ model, whilst alternative explanations receive insufficient consideration.\nMajor Concerns:\n1. Departure from Physiological Context:\nPrior work established that 2t-16, generated by ER lipid metabolism, is shuttled to mitochondria to regulate BAX. Building on these findings, subsequent studies documented a covalent mechanism of regulation for this lipid electrophile. In contrast, the present study departs from the physiologically relevant membrane context by applying high-dose 2t-16 in solution to cells and liposomes (a “direct treatment model” that is insufficiently justified and moves further away from physiological relevance). Further, the membrane environment may facilitate BAX conformations amenable to covalent derivatization, which would not be readily recapitulated by a solution system. The lipid, solubilised using DMSO and prewarming techniques, likely exists as heterogeneous micelles or aggregates, raising concerns regarding its bioavailability and mechanism of intracellular delivery. Intracellular entry beyond plasma membrane incorporation appears doubtful, and any 2t-16 reaching the cytoplasm would likely be rapidly reduced or react with multiple off-target proteins. The observed cellular effects might therefore reflect a non-specific stress response culminating in apoptosis rather than direct BAX regulation.\nFurthermore, the use of serum starvation—a known pro-apoptotic stimulus—as the delivery context for 2t16 further complicates the interpretation. Similar concerns extend to the in vitro data, where the observed effects could more plausibly derive from non-specific detergent-like action, as evidenced by the lack of distinction between wild-type and cysteine-mutant BAX.\n2. Limitations of the Mass Spectrometry Analysis:\nThe MS experiments relied on to discount covalent modification are not sensitive for detecting lipidated BAX due to technical limitations. Lipidated peptides are very hydrophobic and often poorly recovered in MS workflows, and activated BAX oligomers are typically resistant to proteolytic digestion and mass spectrometric detection. Thus, the absence of detectable lipid-modified peptides does not exclude the presence of covalent modifications, particularly given the methodological constraints and the limited experimental detail (e.g., lipid-to-protein ratios, incubation times) provided.\n3. Inappropriate Use of Rigid-Body Docking:\nThe docking studies are performed using the structure of inactive BAX, whereas the proposed binding site is thought to emerge following BIM-induced conformational activation. As such, the docking is based on an inappropriate structural template, and the conclusions drawn are therefore questionable. For a protein as conformationally dynamic as BAX, molecular dynamics simulations would offer a more appropriate approach than rigid-body docking.\n4. Technical Concerns with the NMR Data and Its Interpretation Based on the Structure of Inactive BAX:\nThe interpretation of the NMR data raises additional concerns. First, the data are analyzed in the context of the inactive BAX structure, whereas the proposed model places non-covalent interaction of 2t-16 with BAX downstream of BIM activation. This is problematic because BIM activation is known to induce conformational changes that could alter the proposed binding site, currently mapped to and evaluated based on the inactive state. Second, the timing parameters of the NMR experiments —specifically, the duration of protein-ligand incubation and the time point of NMR data acquisition—are not disclosed, limiting the ability to assess the relevance of the observations to the proposed model. Since BAX activation by 2t-16 promotes dimer (as suggested by the SEC data) and ultimately oligomer formation, such species that could be induced by covalent modification would not be visible by NMR due to signal attenuation, biasing the solution NMR data toward evaluable monomeric species only for the undisclosed conditions that were applied.\n5. Control Compound and Binding Orientation Issues:\nBased on the authors’ non-covalent ligand binding model, there is little mechanistic rationale for why the reduced form (16CHO) would fail to bind and activate BAX, whereas the α,β-unsaturated aldehyde (2t-16) would. Both compounds are featureless hydrophobic chains differing only at the terminal functional group. If the entirety of the binding mechanism was dependent on the presence of the double bond (however unlikely), a key control beyond 16CHO would be the alcohol derivative (trans-2-hexadecen-1-ol), which maintains the double bond but removes the electrophilic aldehyde functionality. Furthermore, the docking poses suggest an unnatural orientation, with the lipid tail (ordinarily embedded within the membrane) proposed to insert into the hydrophobic groove—an orientation inconsistent with membrane biophysics. Instead, a model involving covalent reaction between a cysteine on the BAX surface and a membrane-exposed and reactive lipid head group appears more plausible, as previously detected and validated in the native membrane context.\n6. Interpretation of Mutagenesis Data:\nThe mutagenesis experiments do not conclusively validate the proposed lipid-binding mechanism. Mutations within the putative binding site could alter BAX activation through a range of alternative structural or functional mechanisms. For example, the P186G mutant is known to exhibit reduced activity, but attributing this to impaired lipid binding is speculative without further rigorous characterisation.\nSummary:\nThis manuscript offers interesting insights into potential non-covalent interactions between 2t-16 and BAX that may influence BAX function. However, the evidence provided does not sufficiently support the conclusion that 2t-16 acts predominantly via a non-covalent mechanism. The data appear more consistent with a complementary non-covalent contribution within an overarching covalent regulatory process, particularly in light of established principles of lipid electrophile biology (including diminished activity of the reduced analog) and lipid orientation within membranes.\nThe study would benefit from additional experimentation in membrane systems, a broader exploration of covalent interactions under physiologically relevant conditions, cellular proof of direct non-covalent interaction with BAX, and a more cautious framing of its conclusions.\nFinally, the characterisation of prior work as limited or flawed, whilst employing even more supra-physiological and indirect methodologies in solution, appears unnecessarily dismissive and detracts from the overall scientific narrative. Further, indicating that “structural and molecular mechanisms remain largely unknown” does not accurately reflect the prior literature on 2t-16 and BAX. A more balanced and integrative perspective—that covalent and non-covalent features likely cooperate in BAX regulation by 2t-16—would better reflect the available data and provide a stronger foundation for future studies."}, "author_response": "We greatly appreciate this Reviewer's support of our work and conclusions, even as we provide a divergent model of 2t16-mediated BAX activation than prior publications. While we do strive to make a compelling argument for our findings and model, we do not intend to discount the conclusions of prior publications, instead we propose explanations for how (and when) each model may be relevant to BAX activation mechanisms. We are encouraged that this Reviewer recognizes our efforts in this regard. Responses to the questions and comments are below; we also extensively edited the manuscript to reduce ambiguity and increase clarity as suggested. We appreciate the Reviewer for highlighting these points in the text and we believe that the revised manuscript is much improved.\nComments:\n• Fig. 1B. Why is the control DMSO (for 40 uM) in the SPARKL analysis show such strong signal, more than the ABT 20 uM? This seems concerning.\nThe dark purple line in Figure 1B represents 40 μM 2t16 without cotreatment of ABT-737. As the reviewer notes, 40 μM 2t16 is sufficient to induce some apoptosis as a solo treatment (and is heightened in the presence of ABT-737 – data line with circles), while 20 μM 2t16+ABT‑737 does not induce death in the Bim−/−Bid−/− MEFs. When compared with WT MEFs (Figure 1A), we interpret this decreased sensitivity (at 20 μM 2t16) to the lack of direct activators and therefore an inability of BAX to be activated, even if the anti-apoptotic reservoir is neutralized by ABT‑737. The 40 μM 2t16 treatment is pro-apoptotic in both WT and Bim−/−Bid−/− MEFs (Figures 1A−B) and we interpret this as a condition that can induce BAX activation directly. As we describe later in Figure 1 using LUV permeabilization studies, while high dose 2t16 can induce BAX activation directly, the major effect of 2t16 is observed in contexts where BAX is activated by BIM. Therefore, we believe the cell death studies align with the subsequent permeabilization studies and that the interpretations throughout Figure 1 are consistent.\nIn case this comment arose from misunderstanding our Figure labels, we modified the labels and legends for Figures 1A–C to increase clarity and adjusted the text in the results section (lines 101−120).\n• Line 111. “[we] observed a loss of apoptosis at lower concentrations of ectopic hexadecenal co-treated with ABT-737, suggesting that BAX activation was part of the underlying mechanism (Figure 1B). Finally, we replicated this experiment in Bax−/−Bak−/− DKO MEFs and observed no cell death (Figure 1C). Collectively, these data support the conclusion that hexadecenal induces apoptotic cell death by acting on effector BCL-2 family proteins.”\n--> While there is nothing wrong with these results, I am puzzled at the conclusion. My conclusion is that ABT-737 obviously doesn’t kill Bax−/−Bak−/− DKO.\nThe key comparison is that 40 μM 2t16 (highest dose), which demonstrated cytotoxicity in MEFs expressing BAX (Figures 1A−B), did not induce cell death in the Bax−/−Bak−/− MEFs. Since there is no BAX to be activated by 2t16, the addition of ABT‑737 did not reveal a death response as it had in BAX-expressing cells. We adjusted the text to better articulate this point and draw the reader to the relevant comparisons (lines 101–120).\n• Line 131, Fig. 1F. “Furthermore, the synergy with hexadecenal was observed with mildly activating concentrations of BIM-BH3 as well (Figures 1F, S1C).”\n--> Could is simply be additive? In order to claim synergy, I think the authors need to present the synergistic calculation…\nTo substantiate the use of \"synergy\" within this work, we confirmed a synergistic effect using the Bliss model and added new data panels reporting the Bliss score for our endpoint LUV permeabilization data throughout the manuscript (Figures 1G, 1I, 1K, 2F, 2H, 6H, and 7G).\nAdditionally, since the original submission, we identified a mathematical model to perform non-linear regression data fitting of the LUV permeabilization kinetics, which permits us to extract certain parameters for comparative analysis. In the revised manuscript, we performed data fitting of our LUV experiments (Figures S1D, S2G) and report the maximal rate for comparisons across several concentrations of BIM‑BH3 and 2t16 (Figures 1F, 2H). Details of the synergy determination and data fitting have been added to the Methods section (lines 1027−1033).\nWe thank the reviewer for this suggestion and believe these additional metrics further support our interpretations and strengthen the revised manuscript.\n• Line 152. “We detected peptide fragments covering both cysteines (C62, C126) and observed no alkylation by hexadecenal (m+238.229 Da);”\n--> Is it possible trypsin can’t cleave after the lysine at close proximity to the modification?\nWhile we cannot exclude this possibility, we note that Jarugumilli et al., 2018 also utilized trypsin digestion within their work. Additionally, if the lack of observed m+238 cysteine residues were due solely to a limitation of the technique, the prevalence of iodoacetamide-modified fragments (m+57) would have been noticeably different in the DMSO vs 2t16 samples, which was not observed (these data are now Figure S2C).\nIn the original submission, we utilized \"bottom-up\" HCD LC-MS analysis; however, multiple reviewers have raised questions regarding the potential limitations of the digestion and detection of \"lipidated\" BAX fragments. Therefore, we conducted additional intact mass spectrometry experiments to assess the status of BAX modifications. Due to limitations of sample preparation (e.g., protein concentrations, sample injection, etc.), experiments utilizing wildtype BAX protein did not provide consistent and accurate data. Instead, we utilized oligomerization-deficient BAX mutants (BAXG108V, BAXR109D) to confirm no modification in response to 2t16; as a control, we did observe cysteine modification in response to iodoacetamide treatment (Figures 2B−C; lines 167−174).\nWhile we believe the data from both mass spectrometry approaches are compelling, we agree that they are not conclusive. As such, we corroborated our conclusions by utilizing a cysteine-deficient BAX mutant (BAX2S) in mechanistic and functional assays throughout the study, thereby demonstrating that synergistic activation of BAX by 2t16 is independent of cysteines (Figures 2, 3, 5, S2, S3, and S7). Additionally, we added experiments demonstrating that wildtype BAX modified with iodoacetamide remains sensitive to 2t16 (Figures 2D−F; lines 175−179). Collectively, these diverse approaches all corroborate our conclusion that, at this concentration range, 2t16 results in BAX activation through a non-covalent interaction.\n• The authors report chemical shift changes – but what about comparing line broadening between the mutants? At the residue level, or simply using 1D HN relaxation envelop? There seem to be a loss in chemical shift dispersion (Supp. Fig. 4A)\nWe did observe that several residues peripheral to the BAF (α4−5 loop, α5, α6, α7, α8) exhibited altered peak morphology, such as splitting or merging. We have included this analysis in the revised manuscript (Figure 4A, right panel; lines 267−270).\nIn addition to reporting the chemical shift perturbations (CSPs) for BAX+2t16 (which is calculated from a weighted average of both 1H and 15N shifts) we included an analysis depicting the shifts for each individual dimension (Figure S4C). For the most part, residues exhibiting a significant shift in either the 1H or 15N dimension were also determined to be significant in the CSP calculation, thought this approach did reveal that our analysis may preference towards N shifts.\nThe suggestion to compare NMR results across the BAX mutants is interesting. At present, we have not conducted NMR studies with our BAF mutants. The BAX mutants may exhibit different spectra compared to wildtype and therefore would require substantial effort to reassign the peaks (potentially requiring additional dimensions or analyses). We agree that this could be informative and hope to explore this in future studies.\n• Line 330. I don’t like the term “unbiased docking assessments”. Nothing is unbiased.\nWe have replaced \"unbiased\" with \"unconstrained\" throughout the main text and figure legends, which we agree is a more accurate description of the approach.\n• Line 399. Typo. “Directly comparison”\nThank you for pointing out this typo.\n• Line 475. “We also observed that short-chain alkenals did not activate BAX, which aligns with a prior study that also demonstrated increased reactivity with short-chain alkenals.”\n--> Increased reactivity doesn’t lead to increase BAX activation?\nWe apologize for not explaining this point sufficiently. Cohen et al., 2020 demonstrated that short-chain alkenals were more proficient at modifying BAX compared to long-chain alkenals. Utilizing BAX laddering assays, they demonstrated that high molecular weight species of BAX were formed proportional to alkenal length, with long-chain alkenals exhibiting less monomeric and more oligomeric bands. Our results provide an explanation for this reciprocal relationship between reactivity and BAX activation proportional to alkenal chain length. Our BAX activation assays corroborate that short-chain alkenals fail to activate BAX compared to 2t16, and our structural and in silico approaches suggest that this is due to reduced specificity for the BAF (Figures 5, S5, S6). We have modified the text to better articulate how our model fits with this prior study (lines 375−381).\nReviewer #2:\nThe study by Gelles et al. presents a multidisciplinary investigation into how the lipid metabolite 2t-hexadecenal enhances BAX activation, a central event in mitochondrial apoptosis. The authors combine NMR, computational modeling, biochemical and cellular assays, and mutagenesis to provide mechanistic insight into this interaction. They show that 2t-hexadecenal binds a novel binding region on BAX, a hydrophobic cavity formed by helices α5, α6, and gated by α8, which they term the BAX Actuating Funnel (BAF). Their data suggest that 2t-hexadecenal binds non-covalently at this site, priming BAX for activation, particularly in the presence of the BH3-only activator BIM. Further, they show that α8 helix mobility and alkenal chain length are critical determinants of lipid-mediated BAX activation. The study is thorough and well-controlled, offering a rational model for how 2t-hexadecenal lipid metabolite can modulate BAX conformational dynamics to facilitate BH3-mediated BAX activation leading to mitochondrial outer membrane permeabilization (MOMP).\nOverall, the manuscript offers a novel conceptual model for lipid-mediated BAX activation and a strong case for the biological relevance of the newly identified BAF site, that may be exploited for therapeutic modulation using small molecules. However, I have listed several issues below that should be addressed to strengthen the manuscript and validate the conclusions before publication.\nWe are delighted that this Reviewer supports the nature of our work and its conclusions. As suggested, we incorporated new data, analyses, and discussion points to address all comments and questions. Thank you for the opportunity to improve this study and its communication.\n1. The authors report a dissociation constant (Kd) of 0.182 μM for 2t-hexadecenal binding to BAX (MST assay), yet concentrations >> 6.5–10 μM are used in FLAMBE, liposomal, and cellular assays, including in the presence of BIM BH3. The rationale for this discrepancy should be clearly explained. Have the authors used concentrations near the Kd concentration? Why functional effects require supersaturating conditions?\nThis is a good point. The MST software reported this value as a Kd, but it appears to be largely derived from the inflection point of the dose curve (these data are now Figure S2A). As such, the concentration of the target (BAX) is relevant to this determination. In this assay, we were using 1 nM BAX and so 2t16 was used in 20−40,000 fold excess (to ensure saturation and accurate curve fitting), with the midpoint at ~180 fold excess. In our functional assays (e.g., FLAMBE, LUVs), the concentration of BAX is regularly 100−150 nM and so using 2t16 at 5−50 μM represents approximately 33−500 fold excess, which aligns with MST results. Also of note, the MST experiments were conducted with low-level detergent – 0.5% Tween-20 in the MST buffer to prevent capillary adsorption and 0.002% CHAPS to inhibit BAX activation – and that may also have affected the determination of this value.\nFor these reasons, we avoided putting much emphasis on this value in the text, but we have added a statement to address the above points (lines 151−155). Additionally, we will now label the metric as an EC50 value instead of a Kd, which is likely more accurate and should avoid over-interpretation of the metric. We believe that addressing this comment has clarified the explanation of these data and thank the reviewer for bringing it to our attention.\n2. The authors report a non-covalent interaction between 2t-hexadecenal and BAX. However, prior studies from two independent groups have provided convincing evidence for covalent modification of BAX by 2t-hexadecenal. Did the authors use comparable experimental conditions and mass spectrometry techniques to exclude the possibility of covalent binding in their assays?\nWe did our best to properly address this in our Discussion by providing sufficient consideration and contemplation to explain the divergence in phenotypes, but it is delicate balance advocating for our findings without appearing to disparage the prior works. We describe some of the major differences in experimental conditions below, and modified part of our Discussion to capture some of these points (lines 494–513).\nThe paper that identified modification at C62 (Jarugumilli et al., 2018) utilized an alkyne derivative of 2t16 and subjected their samples to additional chemical agents as part of their click chemistry and pull-down methods. Additionally, their recombinant BAX (Cat #: BAX-6976H, Creative Biomart) is a C-terminal truncated mutant (aa1-171) with two additional His tags, of which the N-terminal tag is 20 residues long. For cell lysates, they subjected their samples to various detergents which may have altered the availability of the cysteine residues (see our response to comment 4).\nThe paper that identified modification at C126 (Cohen et al., 2020) predominantly used 2t16 and other alkenals in millimolar range (0.5–5 mM), which according to their own data induced the chemical modification. The hydrazide labeling of BAX activated to permeabilize LUVs was a very clever approach, but appears to also label activated BAX in LUVs without 2t16 (albeit to a lesser extent), and there was no control demonstrating that BAX-C62A/C126A could not be labeled by the hydrazide. Additionally, some assays utilized auxiliary chemistries and pull-down methods and their 2t16 was solubilized in ethanol instead of DMSO – we do not know if these differences are consequential to the chemistry they describe.\nUltimately, there came a point at which we had to decide whether to exhaustively attempt to replicate these prior studies and identify conditions to rationalize the divergent results, or to focus on keeping conditions as holistic and relevant to the underlying cell biology, and study aspects of BAX biology not covered in the prior works. As such:\n1. We avoided using reactive chemistries as part of our detection methods and instead focused on techniques to study BAX activation.\n2. We did not use millimolar concentrations of 2t16 because we aimed to study its effect on BAX distinct from covalent modifications and at concentrations that better represented a physiological cellular context.\n3. We chose not to incorporate 2t16 directly into liposomes as this had already been shown and may specifically/solely reflect the biology of the oligomeric conformer. Instead, we focused on how 2t16 affects monomeric BAX activation prior to membrane associations. To this end:\n1. We designed experiments to specifically characterize the early steps of the BAX activation continuum, including approaches not utilized in prior works as well as developing new ones (e.g., our FLAMBE assay to specifically monitor \"trigger\"-induced intramolecular rearrangements).\n2. We extended our structure-function insights of BAX by generating several structural mutants and identifying the determinants of the interaction (such as BAF topology and demonstrating that short-chain aldehydes lack targeting specificity).\n3. We explored the hypothesis that the disease-relevant P168 mutation allosterically alters the BAF resulting in the loss-of-function phenotype.\n4. And we attempted to place our discoveries within the larger established literature of the BAX structure-function relationship by detailing how our results connect to a collection of discrete observations (our Discussion).\nWe hope that the take away for the reader is that each work studies distinct biology, and that BAX is potentially regulated both biophysically (interactions with the BAF) and biochemically (cysteine modification) by 2t16 in discrete stages and environments throughout the BAX activation continuum. We modified our Discussion to better highlight this interpretation of both our data and the prior works (lines 494−533, 602−606).\n3. Given the known reactivity of alkenal metabolites, can the authors confirm the purity and structural integrity of the 2t-hexadecenal used in their experiments? Can they provide batch consistency and analytical validation (e.g., NMR, MS) to help rule out variability of the 2t‑hexadecenal as a factor in observed differences compared to previous studies.\nOur 2t16 is purchased from Avanti Polar Lipids and each lot comes with a certificate of analysis and is confirmed to be >99% pure. We prepare our 2t16 in single use aliquots to minimize oxidation or degradation. Though to directly address this question, we performed mass spectrometry on 2t16 and indeed observed a peak corresponding to the correct molecular weight; we have included this vital control (Figure S1A).\n4. The manuscript briefly suggests that NP-40 (a non-ionic detergent) may explain some discrepancies with earlier findings. The authors should elaborate on this point. How might NP-40 influence lipid-protein interactions in lysates or buffers in a way that affects covalent modification?\nAs it specifically pertains to Jarugumilli et al., 2018, we question whether the inclusion of NP‑40 during cell lysis may have affected BAX structure or exposure of the C62 residue. It is well established that common detergents (including NP‑40) induce BAX activation and oligomerization when used above their CMC (e.g., Antonsson et al., 2000). Additionally, C62, which sits within the key BH3 residues of BAX α2, is predominantly core-facing and not likely to be solvent exposed (Suzuki et al., 2000; PDB 1F16). It is a known hallmark that these α2 residues get exposed following BAX activation (Gavathiotis et al., 2010) and are modeled to be cytosolic facing in the activated BAX dimer at the membrane (Czabotar et al., 2013; Bleicken et al., 2014; PDB 4BDU – N.B. C62 is replaced with serine in this structure and α5 is extended to predict C126). Therefore, we posit that NP‑40 may have facilitated certain steps of the BAX activation continuum and that their 2t16 alkyne was targeting an activated conformer of BAX where C62 is more targetable. This may also explain the divergent result in Cohen et al., 2020, where they concluded C126 was the primary target residue.\nWe apologize for not detailing this thought process sufficiently and modified the Discussion to include this explanation (lines 504−509, 523−526). Additionally, we added structural models of the active BAX dimer to support these discussion points, including the recently published structure of oligomeric BAX (Zhang et al., 2025; PDB 9IXU) (Figures S10A−C). We agree that these details are vital to properly articulating our conclusions and believe this addition will greatly improve the Discussion.\n5. The authors note that the BAF cavity is present in previously reported structures of BAX bound to BID BH3 or BAX BH3 domains and the domain-swapped dimers of BAX. The authors should include a comparative structural Figure highlighting the BAF site across these BAX structures for additional clarity and support of this conclusion.\nTo address this excellent suggestion, we added several structures representing domain-swapped BAX dimers bound to various BH3 peptides with their BAF cavities visualized (Figure S10D). Of note, we did slightly modify the text to specifically highlight structures that featured progressive C-terminal truncations, which is a subsequent point we discuss; as such, the structure bound to BAX-BH3 is no longer included as one of the examples (lines 572−575)\nSimilarly, this comment inspired us to include a visualization of the BAF across the ensemble states of the BAX solution structures – namely, monomeric BAX (PDB 1F16) and BAX triggered by BIM (PDB 2K7W) (Figures S4F−G; lines 283−285, 297−300). We agree that these additional visualizations improve the revised manuscript and thank the reviewer for this suggestion.\n6. Figures lack detailed information about the number of replicates and statistical analyses performed. The authors should include this information to Figure legends wherever applicable.\nWe apologize for this oversight and added additional details to our figure legends and methods section where needed.\nReviewer #3:\nGelles et al. investigate the mechanism by which the bioactive sphingolipid metabolite trans-2-hexadecenal (2t16) promotes BAX activation, a process relevant to mitochondrial outer membrane permeabilisation and apoptotic cell death. Employing biochemical, structural, and computational approaches, the authors identify a hydrophobic region of BAX as a putative binding site for non-covalent interaction with 2t16. The authors propose that this interaction, particularly in the presence of the BH3-only activator BIM, potentiates early BAX conformational changes and promotes membrane permeabilisation, without invoking the covalent mechanism elucidated in prior studies.\nWhilst the study employs several creative indirect methods and identifies a potential lipid interaction site, the central claim that 2t16 operates principally through a non-covalent mechanism rests on incomplete and, at times, internally inconsistent evidence. Given the well-established reactivity of α,β-unsaturated aldehydes and the membrane-embedded nature of lipid regulation, much of the data—derived largely from high-concentration solution-phase lipid treatments and rigid-body docking to an inactive BAX structure—appears inconsistent with the proposed model. The findings may ultimately align better with a dual mechanism involving both covalent and non-covalent features, as is commonly observed in physiological and pharmacological covalent protein regulation.\nMoreover, the study is marked by a considerable degree of speculation, with data interpreted selectively to support the authors’ model, whilst alternative explanations receive insufficient consideration.\nWe respect and appreciate this Reviewer's critical analysis of our work and we believe that their perspective may be shared by a subset of the readership. As such, we wish to thoroughly and satisfactorily address the points made below and so we will separate the comments and provide our responses in more granular detail to specific concerns.\nOverall, we believe that several of these comments arose from the Reviewer's interpretation that we contradict and/or dismiss the prior works describing a covalent modification by 2t16. This is undoubtably a consequence of our attempt to defend our findings and a failure to articulate that we suggest a cohesive model in which BAX is likely being regulated both biophysically (interactions with the BAF) and biochemically (cysteine modification) by 2t16 at discrete stages and environments throughout the BAX activation continuum. As we will specifically remark below, we have substantially altered the text of our Discussion to address this.\nOf note, this Reviewer appears to focus heavily on the structure, function, and regulation of oligomeric BAX localized to the mitochondrial outer membrane and several comments suggest less mechanistic attention on BAX biology prior to membrane association. There is ample and growing literature studying monomeric BAX activation and regulation in solution separate from the functionalization phase within a membrane. Here, we specifically focus on the biology and structure of monomeric BAX prior to membrane associations and multimeric structures. We do this in part to distinguish our efforts from prior works while still supporting and leaving space for their conclusions; additionally, it affords focus on complementary yet distinct approaches, questions, and investigations:\n1. We designed experiments to specifically characterize the early steps of the BAX activation continuum, including approaches not utilized in prior works as well as developing new ones (e.g., our FLAMBE assay to specifically monitor \"trigger\"-induced intramolecular rearrangements).\n2. We extended our structure-function insights of BAX by generating several structural mutants and identifying the determinants of the interaction (such as BAF topology and demonstrating that short-chain aldehydes lack targeting specificity).\n3. We explored the hypothesis that the disease-relevant P168 mutation allosterically alters the BAF resulting in the loss-of-function phenotype.\n4. And we attempted to integrate our discoveries within the larger established literature of the BAX structure-function relationship by detailing how our results connect to a collection of discrete observations (our Discussion).\nMajor Concerns:\n1. Departure from Physiological Context:\nPrior work established that 2t16, generated by ER lipid metabolism, is shuttled to mitochondria to regulate BAX. Building on these findings, subsequent studies documented a covalent mechanism of regulation for this lipid electrophile. In contrast, the present study departs from the physiologically relevant membrane context by applying high-dose 2t16 in solution to cells and liposomes (a “direct treatment model” that is insufficiently justified and moves further away from physiological relevance).\nThere is a bit of a misunderstanding regarding the original work identifying the 2t16 requirement. While Chipuk et al., 2012 does detail the requirement of sphingolipids originating in heterotypic membrane populations, the results actually demonstrate that the catabolism of sphingomyelin into ceramide requires ER-localized sphingomyelinases; the subsequent catabolism to 2t16 is accomplished with enzymes found within the mitochondrial outer membrane. The reader may be inclined to restrict their conclusion to a model in which 2t16 must reside in the membrane. However, this is not the only interpretation and several experiments in this original work demonstrate that ectopic 2t16 (i.e., solution-localized) promotes BAX-mediated MOMP and there was no requirement to incorporate 2t16 into model liposomes. While other groups explored the hypothesis that membrane-localized 2t16 interacts with membrane-localized BAX, we chose to explore the open hypothesis that 2t16 interacts with monomeric BAX prior to association with membranes. Moreover, the majority of S1P studies highlight cytosolic and extracellular roles for this lipid and its metabolites, further providing a rationale that 2t16 is not limited to membranes. We have added this note to our text in case any readers similarly question the validity of 2t16 in the cytosol (lines 530−533).\nWe believe that demonstrating a mechanistic role for 2t16 on monomeric conformers of BAX is both physiological and warranted. BAX activation is initiated in the cytosol and it is relevant to investigate how 2t16 may contribute to BAX early-activation distinct from investigations focused on BAX functionalization (e.g., oligomerization and pore formation).\nThe Reviewer critiques our approach as using \"high-dose\" 2t16 when in fact we limit our investigations to low micromolar concentrations. Compare this with prior publications such as Cohen et al., 2020 in which they utilized millimolar concentrations (equivalent to 1000-fold molar excess of BAX), which, as shown in their own data, instigates the covalent modification in solution. Additionally, both Chipuk et al., 2012 and Cohen et al., 2020 demonstrate that millimolar concentrations of 2t16 can induce BAX oligomerization in a manner similar to detergents (i.e., the BAX laddering assays, which require either crosslinking, membranes, or micellar detergents to achieve). While our original submission did use the term \"supraphysiological\" to describe the higher end of our titrations, it was to highlight that direct activation by 2t16 at high concentrations was not likely to occur in cells; however, all doses are in the micromolar range and well-below that of prior publications. To avoid this confusion, we removed that term from our revised manuscript.\nFurther, the membrane environment may facilitate BAX conformations amenable to covalent derivatization, which would not be readily recapitulated by a solution system.\nThis is precisely the point we wish to make in the paper. It is our conclusion that when BAX is activated in the cytosol, the role of 2t16 is mediated by non-covalent interactions within the BAF; at the membrane, we believe the cysteines are more exposed and susceptible to modification by 2t16. Based on the structure of the BAX activated dimer (PDB: 4BDU), C62 is exposed to the cytosol and C126 is likely facing the mitochondrial outer membrane (though the structure does not include C126, the model is extended in Bleicken et al., 2014); the recent BAX oligomer structure further validates this orientation (PDB: 9IXU; Zhang et al., 2025). This model is supported by the compelling LUV data in Cohen et al., 2020 that demonstrates C126 modification when BAX is within the membrane.\nWe acknowledge that we may have not sufficiently articulated this argument or our support for the prior works, and so we have greatly revised the Discussion to better communicate these thoughts as requested by both Reviewer 2 and Reviewer 3 (lines 514−533, 604−606). Additionally, we have included the structures for the active BAX dimer and oligomer, highlighting the orientation of the relevant residues (Figure S10A−C).\nThe lipid, solubilised using DMSO and prewarming techniques, likely exists as heterogeneous micelles or aggregates, raising concerns regarding its bioavailability and mechanism of intracellular delivery. Intracellular entry beyond plasma membrane incorporation appears doubtful, and any 2t16 reaching the cytoplasm would likely be rapidly reduced or react with multiple off-target proteins.\nNumerous publications treating cells with ectopic 2t16 do not support this point. While we cannot eliminate the possibility that 2t16 could form micelles at some critical concentration, 1) the lack of polar or charged region would likely weaken any micelle-forming behavior, thus requiring greater concentrations to induce; and 2) our deliberate avoidance of millimolar concentrations would aid in preventing any micelle or aggregate formation. We agree that any electrophile behavior of 2t16 would certainly target a myriad of cellular targets (as demonstrated in Jarugumilli et al., 2018), and this is precisely why we further interrogate the synergistic mechanism of 2t16 on BIM-activated BAX in additional model systems.\nThe observed cellular effects might therefore reflect a non-specific stress response culminating in apoptosis rather than direct BAX regulation. Furthermore, the use of serum starvation—a known pro-apoptotic stimulus—as the delivery context for 2t16 further complicates the interpretation.\nWe agree that the cellular data can have alternative interpretations and does not de facto prove 2t16 is directly activating BAX. We cannot dismiss the literature demonstrating that ectopic 2t16 can induce stress pathways (e.g., Kumar et al., 2011; Upadhyaya et al., 2012). Nevertheless, we believe that the rapid death kinetics suggest a direct mechanism with BAX; any induced stress response would take several hours to engage the apoptotic machinery – for instance, the time to induce BIM expression and neutralize the anti-apoptotic reservoir. Still, this is precisely why we move away from the cellular system and utilize model systems and techniques to specifically observe and analyze the direct effect of 2t16 on BAX. We have adjusted the text on to make this point more clear (lines 121−127).\nRegarding the point about the serum, we believe that the Reviewer misunderstood our methodology. The cells are not serum-starved. For SPARKL assays, the growth media is removed and replaced by assay media in a two-step process: first, media containing treatments is added, then followed by media containing detection reagents. The treatments (including 2t16, ABT-737, and vehicle controls) were prepared in serum-free media to avoid albumin binding; the detection reagents were prepared in serum-containing media. The cells were first given the media containing treatments, permitted to uptake 2t16 for 30 minutes, and then were given the remaining media, which contained serum. Our controls do not indicate that this approach was detrimental to the cells. We have revised the SPARKL section of our Methods to better detail the experimental setup and avoid this misunderstanding (lines 951–955).\nSimilar concerns extend to the in vitro data, where the observed effects could more plausibly derive from non-specific detergent-like action, as evidenced by the lack of distinction between wild-type and cysteine-mutant BAX.\nThis is only one interpretation of the cysteine-deficient BAX mutant data. Here, we demonstrate no loss of our phenotype when we remove the sites for aldehyde modification, which is a routine approach to investigate post-translational modifications. To view this critical control as somehow indicating a detergent-like behavior appears to be a reluctance to consider any non-covalent mechanism, which is the more straightforward interpretation of these data. We do not agree with the above interpretation for several reasons:\n1. If 2t16 were capable of forming micelles and behaving like a detergent at the micromolar range, then this behavior would similarly call into question the results of Cohen et al., 2020, where it was used at 5 mM.\n2. If the observed BAX phenotypes were due to a non-specific detergent mechanism of 2t16, then we would expect to see an analogous disruption of BCL‑xL/BAKTAMRA in our fluorescence polarization assays, which is not observed (Figure S3H; lines 229–231).\n3. BAX activation by micellar detergent can form oligomers in solution and we do not observe this in our SEC experiments. This is a topic we have previously described in Gelles et al., 2022, and we have added this point to our text (lines 248–250).\n4. In Chipuk et al., 2012, 2t16 at 0.5 and 1 mM demonstrated an ability to directly induce BAX oligomerization in solution-based cross-linking studies. By comparison, the functional assays demonstrated BAX activity with 2t16 in the micromolar range. This example clearly articulates the importance of why we avoided millimolar 2t16 for mechanistic studies.\n5. As stated above, the chemistry of 2t16 and lack of polar or charged region would reduce any micelle-forming or detergent-like behavior, thus increasing the hypothetical critical micellar concentration beyond the range that we utilize it in this work.\nWe do acknowledge that readers may also share this Reviewer's concern and so we have made text adjustments to address the above points (as noted).\n2. Limitations of the Mass Spectrometry Analysis:\nThe MS experiments relied on to discount covalent modification are not sensitive for detecting lipidated BAX due to technical limitations. Lipidated peptides are very hydrophobic and often poorly recovered in MS workflows, and activated BAX oligomers are typically resistant to proteolytic digestion and mass spectrometric detection. Thus, the absence of detectable lipid-modified peptides does not exclude the presence of covalent modifications, particularly given the methodological constraints and the limited experimental detail (e.g., lipid-to-protein ratios, incubation times) provided.\n“[…] activated BAX oligomers are typically resistant to proteolytic digestion and mass spectrometric detection.” This is not supported by the BAX literature. Papers describing altered proteolytic digestion of oligomeric BAX are describing membrane-bound structures of BAX (Goping et al., 1998; Lucken-Ardjomande et al., 2008; Montessuit et al., 2010). Furthermore, Jarugumilli et al., 2018 utilized trypsin digestion to analyze modified BAX by MALDI-TOF.\nHowever, as multiple reviewers have raised questions regarding the potential limitations of the \"bottom-up\" mass spectrometry approach, we conducted additional intact mass spectrometry experiments to assess the status of BAX modifications. Due to limitations of sample preparation (e.g., protein concentrations, sample injection, etc.), experiments utilizing wildtype BAX protein did not provide consistent and accurate data. Instead, we utilized oligomerization-deficient BAX mutants (BAXG108V, BAXR109D) to confirm no modification in response to 2t16; as a control, we did observe cysteine modification in response to iodoacetamide treatment (Figures 2B−C; lines 167−174).\nWhile we believe the data from both mass spectrometry approaches are compelling, we agree that they are not conclusive. As such, we corroborated our conclusions by utilizing a cysteine-deficient BAX mutant (BAX2S) in mechanistic and functional assays throughout the study, thereby demonstrating that synergistic activation of BAX by 2t16 is independent of cysteines (Figures 2, 3, 5, S2, S3, and S7). Additionally, we added experiments demonstrating that wildtype BAX modified with iodoacetamide remains sensitive to 2t16 (Figures 2D−F; lines 175−179). Collectively, these diverse approaches all corroborate our conclusion that, at this concentration range, 2t16 results in BAX activation through a non-covalent interaction.\n3. Inappropriate Use of Rigid-Body Docking:\nThe docking studies are performed using the structure of inactive BAX, whereas the proposed binding site is thought to emerge following BIM-induced conformational activation. As such, the docking is based on an inappropriate structural template, and the conclusions drawn are therefore questionable. For a protein as conformationally dynamic as BAX, molecular dynamics simulations would offer a more appropriate approach than rigid-body docking.\nThe model we describe is one in which α8 is mobilized following BIM triggering and the subsequent accessibility of the BAF permits 2t16 binding. As such, we predominantly utilize the structure of quasi-active BAX bound by BIM-SAHB with α8 removed (PDB 2K7W). We also include in silico investigations utilizing the inactive monomer structure (PDB 1F16) to explore the topography of the BAF in both inactive and triggered monomeric BAX. We are not investigating membrane-associated BAX and therefore do not utilize active dimer structures in our docking studies. We believe we are utilizing the most relevant and appropriate structures for our studies and are curious what structure would be considered more appropriate by this Reviewer.\nWe would like to remind the Reviewer (and future readers) that BAX does not immediately convert from a monomer to the active dimer, and that there is a growing appreciation for distinct conformers along the activation continuum (e.g., loop displacement, α9 mobilization, latch/core separation, etc.). However, these conformers are flexible and mobile, and therefore not conducive to structural biology determinations; at present, there are no structures available to represents these events prior to the active dimer.\nWhile we absolutely agree that molecular dynamics simulations may provide additional insights and could be more informative than rigid body docking, this would represent a significant departure from our current capabilities and would require substantial time to develop. As such, we plan to explore these methodologies in future works and believe that our conclusions are properly supported by the approaches and experiments already included within this work.\n4. Technical Concerns with the NMR Data and Its Interpretation Based on the Structure of Inactive BAX:\nThe interpretation of the NMR data raises additional concerns. First, the data are analyzed in the context of the inactive BAX structure, whereas the proposed model places non-covalent interaction of 2t16 with BAX downstream of BIM activation. This is problematic because BIM activation is known to induce conformational changes that could alter the proposed binding site, currently mapped to and evaluated based on the inactive state. Second, the timing parameters of the NMR experiments—specifically, the duration of protein-ligand incubation and the time point of NMR data acquisition—are not disclosed, limiting the ability to assess the relevance of the observations to the proposed model. Since BAX activation by 2t16 promotes dimer (as suggested by the SEC data) and ultimately oligomer formation, such species that could be induced by covalent modification would not be visible by NMR due to signal attenuation, biasing the solution NMR data toward evaluable monomeric species only for the undisclosed conditions that were applied.\nSimilar to the preceding comment, we are unclear which structure would be considered more appropriate by this Reviewer. BAX protein was treated with 2t16 and subjected to 1H-15N HSQC. Therefore, we utilize the monomeric structure to illustrate the positions of the residues exhibiting significant CSPs (Figure 4A). As the Reviewer states, attempting to capture a BIM-bound \"triggered\" BAX structure by HSQC is extremely complex and typically requires additional detergents and stabilizers to prevent BAX oligomerization during sample collection; for this reason, we studied 2t16 on unstimulated BAX protein. Despite this, we are confident that our viewpoint does not invalidate this experimental approach. The HSQC data demonstrates a congregation of residue shifts around α8, which suggests the location of 2t16 interactions. Moreover, as suggested by Reviewer 1, we have added information regarding the alteration of peak morphology in the residues proximal to α8 (Figure 4A, right panel; lines 267−270). Our interpretation of these data is that this is where 2t16 demonstrates preferential interactions, and we strengthen this conclusion using unconstrained in silco docking (Figure 4C). Furthermore, we confirm α8 mobilization as a requirement for 2t16-induced BAX activation through a disulfide-tethered structural mutant (Figures 4G−I, S4H−M). Critically, while these approaches were what led to our exploration of the BAX cavity, we do not suggest that the observed CSPs represented the binding of 2t16 within the BAF; the interaction residues were further explored in later figures (Figures 6 and 7).\nRegarding the specifics of the sample preparation, we apologize for this omission. Samples were prepared and immediately analyzed by HSQC, which took ~500 minutes to perform the 128 scans at the resolution of complex points we were observing. The length of the collection time is a consequence of the BAX concentration, which cannot be increased without risking auto-activation and precipitation of the sample; this is also consistent with the BAX literature. Therefore, the \"treatment time\" encompasses and surpasses those of our functional assays. We did not observe significant 2t16-induced loss of signal or peak intensities suggestive of protein loss (due to either activation-induced multimerization or degradation). We have adjusted the Methods section to include these details (lines 1192−1206) and added a comment about sample stability to the Results (lines 270−272).\n5. Control Compound and Binding Orientation Issues:\nBased on the authors’ non-covalent ligand binding model, there is little mechanistic rationale for why the reduced form (16CHO) would fail to bind and activate BAX, whereas the α,β-unsaturated aldehyde (2t16) would. Both compounds are featureless hydrophobic chains differing only at the terminal functional group. If the entirety of the binding mechanism was dependent on the presence of the double bond (however unlikely), a key control beyond 16CHO would be the alcohol derivative (trans-2-hexadecen-1-ol), which maintains the double bond but removes the electrophilic aldehyde functionality.\nThis is an important suggestion and we thank the reviewer. While we can replicate the lack of BAX activation with the saturated aldehyde, hexadecanal, we have not yet discovered the underlying mechanism. While we could not source the molecule specifically suggested by the Reviewer (trans‑2‑hexadecen‑1‑ol), we did explore this suggestion in principle. We utilized a hydrazide molecule that would react with the aldehyde head group of 2t16 and observed that the resulting molecule (2t16‑NN‑EtOH) was incapable of activating BAX or synergizing with BIM (Figures 1J−K, S1F; lines 141−143). Later in the paper, we perform in silico docking simulations with 2t16‑NN‑EtOH and observed altered orientations within the BAF due to the new larger headgroup (Figures S8A−B; lines 389−396). These data corroborate our interpretation that the functional consequence of 2t16 is a result of residing in the neck of the BAF, which is lost with 2t16‑NN‑EtOH molecule.\nFurthermore, the docking poses suggest an unnatural orientation, with the lipid tail (ordinarily embedded within the membrane) proposed to insert into the hydrophobic groove—an orientation inconsistent with membrane biophysics. Instead, a model involving covalent reaction between a cysteine on the BAX surface and a membrane-exposed and reactive lipid head group appears more plausible, as previously detected and validated in the native membrane context.\nThe Reviewer is perhaps inadvertently contradicting themselves with this statement, and appears to be confusing the details of prior works. Both Jarugumilli et al., 2018 and Cohen et al., 2020 describe a Michael addition via the β-carbon of the double bond; neither work suggests a Schiff reaction with the aldehyde headgroup. Moreover, neither previous work nor our own implicates the BAX hydrophobic groove (formed by helices α3, α4, and α5). Finally, the lipid-protein overlay assay described in Chipuk et al., 2012 would not have formed a covalent bond and is the most direct example that 2t16 interacts with monomeric BAX structures. Therefore, the model as suggested here by the Reviewer is not supported by the published data.\n“[…] the native membrane context.” Again, we remind the Reviewer that native BAX resides in the cytosol – not the membrane – and that BAX activation occurs in solution prior to translocation to the mitochondrial outer membrane. Our perspective is that regulation of BAX activation occurs predominantly at this stage in the cytosol. Therefore, this work explores the role of 2t16 interactions on monomeric BAX activation prior to translocation, association with membranes, or oligomerization.\nWe agree that the question regarding 2t16 orientation is a good point. All of our molecular docking investigations resulted in a \"tail-first\" orientation of 2t16 in the BAF. We were only able to observe a \"head-first\" orientation if we introduced a constraint forcing the oxygen atom to be in proximity with select BAF residues. Curiously, this did result in a slight benefit to docking score even though the pose could not be replicated in the unconstrained attempts. In light of these results, we are incorporating statements in the Results describing this outcome and that we cannot exclude the possibility of the reverse orientation (lines 397−402). However, as our model describes 2t16 residence in the depth of the BAF as the pro-apoptotic phenomenon – which is supported by the new data with the loss-of-function molecule, 2t16‑NN‑EtOH (Figures 1J−K, S1F, S8A−B; lines 141−143, 389−396) – we do not believe that this open question of orientation undermines the conclusions of this work.\n6. Interpretation of Mutagenesis Data:\nThe mutagenesis experiments do not conclusively validate the proposed lipid-binding mechanism. Mutations within the putative binding site could alter BAX activation through a range of alternative structural or functional mechanisms. For example, the P186G mutant is known to exhibit reduced activity, but attributing this to impaired lipid binding is speculative without further rigorous characterisation.\nWe agree that our mutagenesis approaches may not be 100% conclusive – no singular approach ever is – but collectively, we posit our complementary assays are compelling. This comment is selectively highlighting a known loss-of-function mutant while omitting the relevant controls and data we provide for each of the mutants throughout the paper.\n1. We corroborated the synergistic effect of 2t16 on a cysteine-deficient mutant of BAX (BAX2S) (Figures 2, 3, 5, S2, S3, and S7).\n2. We test our hypothesis that 2t16 binds the BAF through the identification of 3 separate mutants, each of which we validate has having no impaired response to BIM activation or ability to permeabilize LUVs (Figures S8C−D).\n3. Regarding P168G, we explicitly comment that this mutant has reduced activity (lines 454−456) and demonstrate the reduced, but not abrogated, response to BIM and ability to permeabilize LUVs (Figures S9B).\n4. In the revised manuscript, we now include Bliss synergy calculations to quantitatively determine that the BAF mutants exhibit loss of sensitivity to 2t16-induced synergy (Figure 6H).\n5. In the case of P168G, the Bliss calculations quantify the loss of 2t16 synergy while accounting for the reduced sensitivity to BIM (Figure 7F−G).\n6. We specifically study the loss of 2t16 sensitivity on P168G in our BAX activation assays (e.g., FLAMBE, LUV permeabilization) prior to experimentation in cellular models in order to contextualize the results from cellular model systems.\nWhile we agree with this Reviewer that the loss of sensitivity to 2t16 in reconstituted double knockout MEFs could be the result of a general reduced activity of the P168G mutant, this is why we first assessed the response of P168G to 2t16 in our BAX activation assays (Figures 7D−G). We have adjusted the language of our results section to avoid unwarranted overinterpretation of the results (lines 463−467).\nSummary:\nThis manuscript offers interesting insights into potential non-covalent interactions between 2t16 and BAX that may influence BAX function. However, the evidence provided does not sufficiently support the conclusion that 2t16 acts predominantly via a non-covalent mechanism. The data appear more consistent with a complementary non-covalent contribution within an overarching covalent regulatory process, particularly in light of established principles of lipid electrophile biology (including diminished activity of the reduced analog) and lipid orientation within membranes.\nThe study would benefit from additional experimentation in membrane systems, a broader exploration of covalent interactions under physiologically relevant conditions, cellular proof of direct non-covalent interaction with BAX, and a more cautious framing of its conclusions.\nFinally, the characterisation of prior work as limited or flawed, whilst employing even more supra-physiological and indirect methodologies in solution, appears unnecessarily dismissive and detracts from the overall scientific narrative. Further, indicating that “structural and molecular mechanisms remain largely unknown” does not accurately reflect the prior literature on 2t16 and BAX. A more balanced and integrative perspective—that covalent and non-covalent features likely cooperate in BAX regulation by 2t-16—would better reflect the available data and provide a stronger foundation for future studies.\nWe greatly regret and apologize if our original submission was interpreted as dismissive of prior works. This was not our intention, and we may have been a bit overzealous while advocating for our findings and providing distinction between our approach and the approaches of others. We have adjusted our language throughout the manuscript, with particular attention to sections remarking on the other works (lines 494−533). Though as requested by Reviewer 2, we do include commentary on how the different approaches may have resulted in divergent conclusions; however, the language has been adjusted to avoid a negative or dismissive interpretation. (lines 494−513). Similarly, we have adjusted the language in our introduction to better articulate what remains \"an open question\" (lines 84−86).\nTo be clear and reiterate: we believe that each work studies distinct biology, and that BAX is likely being regulated both biophysically (interactions with the BAF) and biochemically (cysteine modification) by 2t16 in discrete stages and environments throughout the BAX activation continuum. It is our conclusion that when BAX is activated in the cytosol, the role of 2t16 is mediated by non-covalent interactions within the BAF; at the membrane, we believe the cysteines are more exposed and susceptible to modification by 2t16, which is supported by structures of BAX at the membrane. We have reworked our Discussion to explore this cohesive model more thoroughly, as requested by both Reviewer 2 and Reviewer 3 (lines 523−529, 602−606), and included relevant structures of active multimeric BAX to support this more inclusive conclusion (Figure S10A−C).\nWe respectfully request that the Reviewer re-evaluates if our defined and faithful model systems soundly represent a means to study the BAX activation continuum and its relevant physiological biology – model systems that established and support the majority of what we currently know about pro-apoptotic BCL-2 proteins. Again,\n1. BAX is activated and regulated in the cytosol, and therefore studying BAX prior to membrane associations is valid and warranted.\n2. 2t16 is a soluble long-chain aldehyde and not restricted only to membranes.\n3. Chipuk et al., 2012 included several studies suggestive of interaction between 2t16 and BAX prior to membrane association or oligomerization.\n4. While millimolar concentrations of 2t16 exhibit covalent binding against BAX and a myriad of cellular substrates, the cellular concentration is orders of magnitude lower; we therefore utilize concentrations that are more representative of the cellular environment (low micromolar).\nWe believe that we represent a fundamentally different philosophy of BAX biology compared with this Reviewer, and therefore it appears we have differing opinions on how best to study BAX – and this is ok! We may not share the same perspective, but we hope that our revised manuscript and this thorough response document help explain our position and approach, and that we can respectfully agree to disagree on a few points.\nThat being said, we absolutely agree with the tone check and thank this Reviewer for pushing us to prepare a more cohesive, comprehensive, and inclusive paper, and we believe the revised Discussion is much improved.\nReferences\nAntonsson B, Montessuit S, Lauper S, Eskes R, Martinou JC. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J. 2000 Jan 15;345 Pt 2(Pt 2):271-8. PMID: 10620504; PMCID: PMC1220756.\nBleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, García-Sáez AJ, Bordignon E. Structural model of active Bax at the membrane. Mol Cell. 2014 Nov 20;56(4):496-505. doi: 10.1016/j.molcel.2014.09.022. Epub 2014 Oct 30. PMID: 25458844; PMCID: PMC4869853.\nBleicken S, Assafa TE, Stegmueller C, Wittig A, Garcia-Saez AJ, Bordignon E. Topology of active, membrane-embedded Bax in the context of a toroidal pore. Cell Death Differ. 2018 Nov;25(10):1717-1731. doi: 10.1038/s41418-018-0184-6. Epub 2018 Sep 5. PMID: 30185826; PMCID: PMC6180131.\nChipuk JE, McStay GP, Bharti A, Kuwana T, Clarke CJ, Siskind LJ, Obeid LM, Green DR. Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell. 2012 Mar 2;148(5):988-1000. doi: 10.1016/j.cell.2012.01.038. PMID: 22385963; PMCID: PMC3506012.\nCohen DT, Wales TE, McHenry MW, Engen JR, Walensky LD. Site-Dependent Cysteine Lipidation Potentiates the Activation of Proapoptotic BAX. Cell Rep. 2020 Mar 10;30(10):3229-3239.e6. doi: 10.1016/j.celrep.2020.02.057. PMID: 32160532; PMCID: PMC7343539.\nCzabotar PE, Westphal D, Dewson G, Ma S, Hockings C, Fairlie WD, Lee EF, Yao S, Robin AY, Smith BJ, Huang DC, Kluck RM, Adams JM, Colman PM. Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell. 2013 Jan 31;152(3):519-31. doi: 10.1016/j.cell.2012.12.031. PMID: 23374347.\nGavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol Cell. 2010 Nov 12;40(3):481-92. doi: 10.1016/j.molcel.2010.10.019. PMID: 21070973; PMCID: PMC3050027.\nGelles JD, Mohammed JN, Chen Y, Sebastian TM, Chipuk JE. A kinetic fluorescence polarization ligand assay for monitoring BAX early activation. Cell Rep Methods. 2022 Mar 28;2(3):100174. doi: 10.1016/j.crmeth.2022.100174. Epub 2022 Mar 9. PMID: 35419554; PMCID: PMC9004659.\nGoping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, Korsmeyer SJ, Shore GC. Regulated targeting of BAX to mitochondria. J Cell Biol. 1998 Oct 5;143(1):207-15. doi: 10.1083/jcb.143.1.207. PMID: 9763432; PMCID: PMC2132805.\nJarugumilli GK, Choi JR, Chan P, Yu M, Sun Y, Chen B, Niu J, DeRan M, Zheng B, Zoeller R, Lin C, Wu X. Chemical Probe to Identify the Cellular Targets of the Reactive Lipid Metabolite 2-trans-Hexadecenal. ACS Chem Biol. 2018 May 18;13(5):1130-1136. doi: 10.1021/acschembio.7b01063. Epub 2018 Apr 6. PMID: 29608264; PMCID: PMC5959771.\nKumar A, Byun HS, Bittman R, Saba JD. The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in a JNK-dependent manner. Cell Signal. 2011 Jul;23(7):1144-52. doi: 10.1016/j.cellsig.2011.02.009. Epub 2011 Mar 6. Erratum in: Cell Signal. 2012 Jan;24(1):351. PMID: 21385609; PMCID: PMC3086202.\nLucken-Ardjomande S, Montessuit S, Martinou JC. Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes. Cell Death Differ. 2008 Mar;15(3):484-93. doi: 10.1038/sj.cdd.4402280. Epub 2007 Dec 14. PMID: 18084240.\nMontessuit S, Somasekharan SP, Terrones O, Lucken-Ardjomande S, Herzig S, Schwarzenbacher R, Manstein DJ, Bossy-Wetzel E, Basañez G, Meda P, Martinou JC. Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization. Cell. 2010 Sep 17;142(6):889-901. doi: 10.1016/j.cell.2010.08.017. PMID: 20850011; PMCID: PMC4115189.\nSuzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000 Nov 10;103(4):645-54. doi: 10.1016/s0092-8674(00)00167-7. PMID: 11106734.\nUpadhyaya P, Kumar A, Byun HS, Bittman R, Saba JD, Hecht SS. The sphingolipid degradation product trans-2-hexadecenal forms adducts with DNA. Biochem Biophys Res Commun. 2012 Jul 20;424(1):18-21. doi: 10.1016/j.bbrc.2012.06.012. Epub 2012 Jun 19. PMID: 22727907; PMCID: PMC3402648.\nZhang Y, Tian L, Huang G, Ge X, Kong F, Wang P, Xu Y, Shi Y. Structural basis of BAX pore formation. Science. 2025 Jun 26;388(6754):eadv4314. doi: 10.1126/science.adv4314. Epub 2025 Jun 26. PMID: 40570108."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments adequately.", "reviewer_2": "The authors have satisfactorily addressed my comments with new data, clarifications, and revisions of the text.\nThey investigated two BAX mutants with reduced oligomerization capacity for evidence of covalent adducts formed by 2t-hexadecenal binding and evaluated the BAX-2S mutant (C62S/C126S), which lacks reactive cysteines and therefore cannot form covalent adducts. These new experiments and data strengthen the authors' conclusions and underscore a role for 2t-hexadecenal in mediating early BAX activation in the cytosol. This is also consistent with the structures of BAX conformations available. While prior studies demonstrated covalent lipid adduction to BAX, the present work offers an alternative and potentially complementary mechanism for 2t-hexadecenal. The authors have clearly articulated the study’s conclusions and limitations in the revised manuscript and reviewer's responses. Overall, this is a coherent and well-executed study and I recommend publication.", "reviewer_3": "I appreciate the revision efforts and the clarifying remarks with respect to rationale and methodology. The work introduces interesting tools and concepts regarding early BAX activation, but several interpretive and methodological aspects remain overstated and highly speculative relative to the data.\n1. Positioning of prior work. It would be appropriate to acknowledge up front that covalent modification of BAX by 2t-hexadecenal (2t16) at Cys126 has already been demonstrated in membrane and cellular contexts with functional consequences for MOMP (as opposed to indicating in the abstract and introduction that the molecular mechanism involving 2t16 is “largely unknown” and “remains an open question”). Introducing and acknowledging prior work does not undermine the current work. In accordance with this handling, the discussion concentrates chiefly on the in vitro concentrations used in earlier covalent studies whilst overlooking the accompanying physiological findings that validated these mechanisms in native mitochondria and intact cells in a cysteine-specific manner. The selective emphasis on mM dosing in prior in vitro studies gives an incomplete picture and may inadvertently mislead readers regarding the existing mechanistic evidence and sets a tone that authors are trying hard to “sell” their current work as a controversial alternative. It would be more objective and balanced to frame the present work as exploring non-covalent contributions that complement covalent mechanisms already confirmed in physiologic settings.\n2. Plausibility of cytosolic delivery. The proposal that exogenously applied 2t16 freely enters the cytosol and engages BAX to account for the observed results remains speculative and unlikely – and the authors provide no evidence that lipid-BAX binding in the cytosol is actually occurring (nor that 2t16 even reached the cytosol in reacted or unreacted form). It is so well known that α,β-unsaturated aldehydes are highly reactive electrophiles which, if present in the media and if even able to dislodge from the cell membrane to access the cytosol, would be expected to form adducts with numerous nucleophilic targets, including glutathione and abundant soluble proteins (the authors refer to hundreds of potential reactive targets). In this light, a BAX-related effect to explain the Figure 1A-B data appears so implausible as to undermine the subsequent structure-function work. Starting the manuscript on such thin ice (as in, this is our interpretation yet here are the multitude of caveats that more likely make it not a reliable interpretation) is undermining. The early data is among the weakest data (lack of actual dose-responses, many alternative interpretations, etc.).\n3. Clarification regarding lipid orientation and membrane context. In the response to point 5, the authors appear to have misunderstood an important aspect of the critique. The concern was not about Schiff-base formation with an aldehyde given the Michael addition mechanism, but instead about lipid orientation: the suggestion that the lipid tail of 2t16 inserts into the hydrophobic cleft of BAX (the hydrophobic BAF site not the BC groove) is difficult to reconcile with the disposition of endogenous ER-to-mitochondrial transfer of these lipids, whose hydrophobic tails are embedded within or associated with membranes. A soluble, tail-inserted lipid electrophile acting on cytosolic BAX is conceptually inconsistent with known membrane biology. I appreciate that the authors believe that perhaps 2t16 can find its way to the solution phase, but they never test or show it by fractionation MS or otherwise. In addition, there is no data presented from cells or cell lysates that show this lipid is associated with soluble BAX. Such results are so fundamental to the study – so proceeding without them makes this a really hard sell.\n4. Non-covalent versus covalent framing. The failure of non-reactive analogues (saturated or hydrazide-modified aldehydes) to activate BAX supports the relevance of the α,β-unsaturated moiety to a covalent mechanism, not a non-covalent binding mechanism. The reader will know this, so to interpret this result otherwise is inconsistent with biochemical principles. Along these lines, the cysteine-mutant data do not disprove a covalent mechanism, they only allow for study of an isolated non-covalent contribution. Therefore, the result section subtitles and interpretations in the abstract and conclusion should emphasize the study of a non-covalent contribution rather than position the results as proving a non-covalent only mechanism, which the data simply do not support (and more often suggest the opposite). The NMR data show that the non-reactive 16CHO compound causes NMR CSPs, which would be expected as the non-covalent component of a covalent process, because the specificity of physiologic covalent reactivity and signaling relies on non-covalent interaction to template and reinforce selectivity of the covalent component of the interaction.\n5. Mass-spectrometry limitations and docking language. The authors’ negative MS data do not exclude covalent modification (particularly since two independent groups demonstrated otherwise), and the docking studies would be better presented as hypothesis-generating rather than structural evidence. Since MD simulations are not performed, “docking simulations” terminology should be changed to “rigid-body docking” or “static docking calculations”.\n6. Clarifying the dosing context. The authors emphasize that they are using micromolar rather than millimolar dosing for in vitro studies, but in the response the authors also indicate that in one set of experiments the molar excess is up to 40,000-fold compared to BAX, which undermines the argument that this study is more conservative or physiologic with respect to dosing levels compared to prior ones. The in vitro setting is just that – it is non-physiologic by definition and is used to hypothesis generate for ultimate testing and validation in cells and tissues.\n7. Lingering structural methodology concerns. The manuscript remains heavily reliant on static docking calculations, which constitute a minimal and low-rigor approach given the central role of computation in their conclusions. While the authors argue that molecular dynamics (MD) simulations are beyond scope, this is unpersuasive; MD is precisely the method needed to capture conformational variability, address structural perturbations (e.g., α8 deletion), and evaluate stability of binding modes. The current strategy does not adequately represent the relevant ensembles or intermediates, and the terminology conflating docking with simulation is misleading. At minimum, the authors should employ explicit solvent MD sampling of existing structures, relax mutant models, and dock to representative ensembles. Without this, the work does not meet basic standards for a high-impact publication.\nTo summarize, the authors work so hard at justifying the new model that they undermine their credibility by making comparisons and justifications that aren’t objectively effective or necessary based on the data provided. Making the suggested textual refinements, with an overall more objective approach to data interpretation would allow the manuscript to stand as a more constructive extension of previous models rather than a competing reinterpretation, whilst fully crediting established physiological data and situating the proposed mechanism within the known biochemistry of lipid electrophiles. It may be \"OK\" to directly say, this is what we speculate based on our results despite the caveats, some of our own data (and prior data) are inconsistent with our new model, and even quite inconsistent with what the reader might expect from lipid electrophiles and how they are used in the study. At the end of the day, a more circumspect handling seems not only justified, but could ultimately come across more genuine and compelling."}, "author_response": "We thank the reviewer for appreciating our revision efforts and clarifying remarks, and for the encouraging perspectives on our experimental tools and concepts. We apologize that our first resubmission was not satisfactory for this Reviewer, and are pleased to have the opportunity to improve the manuscript in a second round of revision. Below, we provide detailed responses to the Reviewer’s lingering concerns, and hope that this persuades the Reviewer to agree with Reviewers 1 and 2 that our conclusions are properly supported by the approaches, experiments, and controls included within this work.\n1. Positioning of prior work.\nIt would be appropriate to acknowledge up front that covalent modification of BAX by 2t-hexadecenal (2t16) at Cys126 has already been demonstrated in membrane and cellular contexts with functional consequences for MOMP (as opposed to indicating in the abstract and introduction that the molecular mechanism involving 2t16 is “largely unknown” and “remains an open question”). Introducing and acknowledging prior work does not undermine the current work. In accordance with this handling, the discussion concentrates chiefly on the in vitro concentrations used in earlier covalent studies whilst overlooking the accompanying physiological findings that validated these mechanisms in native mitochondria and intact cells in a cysteine-specific manner. The selective emphasis on mM dosing in prior in vitro studies gives an incomplete picture and may inadvertently mislead readers regarding the existing mechanistic evidence and sets a tone that authors are trying hard to “sell” their current work as a controversial alternative. It would be more objective and balanced to frame the present work as exploring non-covalent contributions that complement covalent mechanisms already confirmed in physiologic settings.\nWe genuinely appreciate the reviewer’s supportive position for distinct, yet potentially, complementary activities of 2t16 on BAX. Therefore, we agree that updating the language to align with the above statements is an excellent suggestion. We edited the Abstract and Introduction (specifically, lines 84–88) to capture these concepts and introduce the prior works earlier in the manuscript, and we further honed the Discussion to include additional interpretations from the previous publications. However, we do believe it is accurate to state that the role of 2t16 on monomeric BAX activation, distinct from membrane-associated multimers, remains an open question worthy of further investigation as it has not been decisively studied by prior works.\nAs an aside, the physiological findings referred to above are more biochemistry leaning than cell biology and/or physiological as no interactions between endogenous BAX and endogenous 2t16 were published. The use of “native mitochondria” (i.e., isolated mitochondria) was an extension of the original study we published in Cell where recombinant BAX was treated with exogenous 2t16 or recombinant S1P lyase in the presence of BIM peptide. It is worth noting that the protocol used for isolating mitochondria also includes heterotypic membranes (this was the premise of the original Cell publication). Finally, the \"emphasis on mM dosing\" is important to include as (we believe) it provides the nuance necessary to explain the different observations between the works. Indeed, Reviewer 2 specifically requested this additional commentary on experimental details that may have resulted in the distinct phenotypes, and we agree that this is valuable to the readership who may otherwise believe that the mechanism of 2t16 on BAX activation is fully described. Furthermore, highlighting the role of substrate concentration in a proposed chemical reaction is entirely warranted and relevant. While this Reviewer feels these details are disparaging in nature, we believe they are constructive to the larger discourse and we explicitly conclude this discussion by positing that both non-covalent and covalent contributions of 2t16 are biologically relevant to BAX activation (lines 516–535, 606–608).\n2. Plausibility of cytosolic delivery.\nThe proposal that exogenously applied 2t16 freely enters the cytosol and engages BAX to account for the observed results remains speculative and unlikely – and the authors provide no evidence that lipid-BAX binding in the cytosol is actually occurring (nor that 2t16 even reached the cytosol in reacted or unreacted form). It is so well known that α,β-unsaturated aldehydes are highly reactive electrophiles which, if present in the media and if even able to dislodge from the cell membrane to access the cytosol, would be expected to form adducts with numerous nucleophilic targets, including glutathione and abundant soluble proteins (the authors refer to hundreds of potential reactive targets). In this light, a BAX-related effect to explain the Figure 1A-B data appears so implausible as to undermine the subsequent structure-function work. Starting the manuscript on such thin ice (as in, this is our interpretation yet here are the multitude of caveats that more likely make it not a reliable interpretation) is undermining. The early data is among the weakest data (lack of actual dose-responses, many alternative interpretations, etc.).\nTo briefly summarize Figures 1A–C, we generated kinetic cellular data suggesting that 2t16-dependent cell death requires BAX and BIM, and this phenotype is regulated by anti-apoptotic BCL-2 proteins. Importantly, our stated conclusion is broad and only suggests that 2t16 \"induces apoptotic cell death by acting on effector BCL-2 family proteins\", but we have further softened the language to avoid overinterpretation (lines 121–122).\nImmediately after describing these observations, we stated, “There are reports that ectopic hexadecenal can form adducts with DNA and generate oxidative stress resulting in apoptosis, and we could not eliminate the possibility that ectopic hexadecenal was targeting additional substrates or stress pathways. Therefore, we interrogated whether hexadecenal directly promoted BAX-mediated pore formation by utilizing recombinant BAX protein and large unilamellar vesicles (LUVs), which are biochemically-defined liposomes that mimic the major lipid composition of the OMM, and assessed BAX activation by measuring LUV permeabilization” (lines 123–129). Within the previous revised document, we fully acknowledged that alternatives exist, and we focused our investigations into a singular pathway to explore BAX biology. In the remainder of the revised manuscript, we then identified and linked direct biochemical, biophysical, and structural relationships between 2t16 and BAX. Given the word limitations for the journal, we cannot exhaustively discuss experimental limitations of the work at each figure panel. We propose that this approach is no different from adding a growth factor to a cell and exploring one node of a signaling pathway or cellular phenotype in isolation; there is no purpose in overly undermining the work with copious alternative interpretations within the Results section. As such, we believe that the progression of our experimental approaches and results is the most appropriate and effective order to thoroughly characterize and report our findings.\nThe comment that “…exogenously applied 2t16 freely enters the cytosol…” is the Reviewer’s personal synthesis, which is not based on our data, interpretation, or discussion. In the 2t16 literature, it is known that the aldehyde forms adducts (albeit potentially reversible) with glutathione and serum albumin, yet it is commonly added to cells to study its activities (the doses utilized account for the proportion binding to glutathione, for example). Similarly, we noted that lowering the serum aided in the cellular response to 2t16. As the focus of the current work is not the mechanism of 2t16 uptake, we did not mention proposed uptake scenarios because it is a frequently used approach to study 2t16; however, this would be interesting biology to pursue, and would likely include investigations of endocytosis, micropinocytosis, and carrier proteins. Furthermore, the sphingolipid field’s early work in the 1990s that identified the first intersections with cell death were based on extracellular/ectopic delivery of numerous amphipathic reactive species, which also served as the foundation for our understanding of how extracellular sphingolipid gradients activate GPCRs and inform the immune compartment.\nFinally, in contrast to the Reviewer’s note, we indeed included multiple 2t16 doses in all cellular experiments (Figures 1A–C, 7G–I) and extensively throughout the approach to ensure scientific rigor.\n3. Clarification regarding lipid orientation and membrane context.\nIn the response to point 5, the authors appear to have misunderstood an important aspect of the critique. The concern was not about Schiff-base formation with an aldehyde given the Michael addition mechanism, but instead about lipid orientation: the suggestion that the lipid tail of 2t16 inserts into the hydrophobic cleft of BAX (the hydrophobic BAF site not the BC groove) is difficult to reconcile with the disposition of endogenous ER-to-mitochondrial transfer of these lipids, whose hydrophobic tails are embedded within or associated with membranes. A soluble, tail-inserted lipid electrophile acting on cytosolic BAX is conceptually inconsistent with known membrane biology. I appreciate that the authors believe that perhaps 2t16 can find its way to the solution phase, but they never test or show it by fractionation MS or otherwise. In addition, there is no data presented from cells or cell lysates that show this lipid is associated with soluble BAX. Such results are so fundamental to the study – so proceeding without them makes this a really hard sell.\nAt present, there is no generally accepted mechanism for how lipid orientation or membrane contexts support S1P metabolism into 2t16 nor release of 2t16 from a hydrophobic environment. The S1P lyase active site faces the cytosol, suggesting that the catalysis of S1P occurs in solution and in trans-membrane structures. For 2t16 metabolism by FALDH, there are implications of enzyme-membrane juxtaposition, 2t16 “sliding” along an ER membrane to enter at the cytosolic/membrane protein interface, and potentially 2t16 transient binding to/from glutathione, but none are uncontested as to how the active site accesses 2t16 prior to catalysis. Additionally, the Reviewer's assumptions regarding the localization and suspected membrane orientation of 2t16 is based on behavior of bulky and complex amphipathic lipids (e.g., phospholipids, glycerolipids) and does not reflect that 1) S1P and α,β-unsaturated aldehydes are soluble moieties and 2) they target cellular substrates that are not associated with membranes. As there is no “consistent with membrane biology” literature to explain how 2t16 orientation supports its metabolism, this is clearly an important area of investigation for the future, but this knowledge gap should not hinder the present study as we provide an additional premise of protein biochemistry to better examine 2t16 metabolism and its consequences.\nDespite this, we addressed this comment during the prior round of revision by: 1) introducing additional constraints to our molecular docking to generate a \"head-first\" orientation (though we were not able to replicate this result without the forced constraints); and 2) adding a section to the Results describing the additional modeling and explicitly stating that the orientation of 2t16 could not be conclusively determined (lines 399–404). Aside from attempting protein crystallography of 2t16 and BAX (which involves significant technical hurdles and limitations), we are unclear how else this comment could be addressed. As such, we provide a rational interpretation based on this literature and in silico tools to glean interpretations, but we clearly indicate in our manuscript that this mechanism is not defined.\nThe reviewer indicates several times that the cytosolic interaction between 2t16 and BAX has been convincingly published (which was originally implicated by our functional data in Cell using isolated primary mitochondria from fresh liver and endogenous S1P lyase). We reproduce this interaction in vitro and extended with new insights into a binding domain and functional consequences – also with a disease related BAX mutant. Regarding the use of cell lysates to observe the complex, there is currently no approach to detect endogenous 2t16; all published methods utilized exogenous and derivatized 2t16 to measure S1P lyase activity. These approaches significantly change the shape of 2t16 and involve multiple organics that exclude detecting a protein complex. Furthermore, we remind the Reviewer that we propose a non-covalent interaction between 2t16 and BAX, and, as such, attempts to isolate cellular BAX would not preserve this interaction.\n4. Non-covalent versus covalent framing.\nThe failure of non-reactive analogues (saturated or hydrazide-modified aldehydes) to activate BAX supports the relevance of the α,β-unsaturated moiety to a covalent mechanism, not a non-covalent binding mechanism. The reader will know this, so to interpret this result otherwise is inconsistent with biochemical principles. Along these lines, the cysteine-mutant data do not disprove a covalent mechanism, they only allow for study of an isolated non-covalent contribution. Therefore, the result section subtitles and interpretations in the abstract and conclusion should emphasize the study of a non-covalent contribution rather than position the results as proving a non-covalent only mechanism, which the data simply do not support (and more often suggest the opposite). The NMR data show that the non-reactive 16CHO compound causes NMR CSPs, which would be expected as the non-covalent component of a covalent process, because the specificity of physiologic covalent reactivity and signaling relies on non-covalent interaction to template and reinforce selectivity of the covalent component of the interaction.\nWe would like to point out that the non-reactive hydrazide product retains the reactive trans-double bond (a Michael addition as detailed in both Jarugumilli et al., 2018 and Cohen et al., 2020), so the suggestion that the hydrazide reaction supports covalent reaction is inconsistent with the experimental design, results, and scientific literature. We also disagree with this Reviewer's extremely selective interpretation of the NMR data and feel compelled to remind them that, in addition to 16CHO demonstrating distinct CSP patterns, short-chain aldehydes (which retain reactivity for cysteine residues via the trans-double bond) similarly demonstrated altered CSP profiles. Critically, neither cysteine residue demonstrated significant CSPs in response to 2t16, so the Reviewer's proposed explanation of the NMR data is not supported by our data.\nTo reiterate our position on covalent versus non-covalent mechanisms (as discussed in response to other comments in this document, and in the previous revision cycle): we are not denying the covalent mechanism, but given cysteine-free monomeric BAX cooperates with BIM and 2t16 to activate and permeabilize membranes, this suggests that alternative, non-covalent mechanisms warrant exploration and these data should be available to the scientific community. Our section subtitles and conclusion text appropriately reflect that our data support (we do not use the term \"prove\") the non-covalent mechanism, and we provide ample space and consideration in the manuscript to describing the relevance of the covalent mechanism as well (lines 516–535, 606–608). We have made multiple additional adjustments to the text to ensure that the reader does not interpret our conclusions as discounting the existence of a covalent mechanism (for example, lines 164–165, 186–188, 482–485).\nAs a larger point, numerous mechanisms explaining several fundamentals within the apoptotic cascade have driven our field for decades. For example, the decade-long disagreement regarding the role of BH3-only proteins in BAX/BAK activation, which ultimately revealed the direct activator and sensitize/de-repressor BCL-2 classes; or the ongoing 20+ year debate as to whether or not the BH3-only protein PUMA is a direct activator. Similar to these examples, previous literature on 2t16/BAX and our current manuscript will support scientific engagement and the establishment of elegant model systems to explore the interactions between the entire BCL-2 family and lipids, in which very little is known. We hope the reviewer appreciates the critical importance of the scientific literature to not only reinforce past work but also challenge and evolve.\n5. Mass-spectrometry limitations and docking language.\nThe authors’ negative MS data do not exclude covalent modification (particularly since two independent groups demonstrated otherwise), and the docking studies would be better presented as hypothesis-generating rather than structural evidence. Since MD simulations are not performed, “docking simulations” terminology should be changed to “rigid-body docking” or “static docking calculations”.\nThe Reviewer appears to interpret our revised manuscript as continuing to argue against and invalidate prior works, which we simply do not, as based on our Introduction and Discussion sections. \"The authors' negative MS data do not exclude covalent modification...\", \"the cysteine-mutant data do not disprove a covalent mechanism...\" – we use these approaches to demonstrate that our observed phenotype is not due to covalent modification, and we explicitly incorporate the conclusions of the prior works in our interpretations and discussion. These comments were raised in the previous revision cycle, and our response to these concerns are maintained in the re-revised manuscript (lines 160–164, 186–188, 480–482, 516–521, 525–531, 606–608). It is unrealistic to include a qualifying statement regarding the prior works for every single panel of data that indicates we are studying a non-covalent mechanism throughout the Results section; instead, the Discussion is the appropriate section of the paper to compare, contrast, and consider these differences.\nFurthermore, we do indeed use the docking studies as hypothesis generating and then experimentally examine the in silico data with several structural mutants (both non-functional and functional) using numerous experimental model systems (Figures 4, 6, 7, S4, S8, S9). For clarity, we doubled checked and refined the language to ensure our scientific process is more obvious to the reader.\nFinally, we agree that since MD simulations are not performed, the terminology was updated to remove any erroneous use of the term \"simulation\". Thank you for this correction.\n6. Clarifying the dosing context.\nThe authors emphasize that they are using micromolar rather than millimolar dosing for in vitro studies, but in the response the authors also indicate that in one set of experiments the molar excess is up to 40,000-fold compared to BAX, which undermines the argument that this study is more conservative or physiologic with respect to dosing levels compared to prior ones. The in vitro setting is just that – it is non-physiologic by definition and is used to hypothesis generate for ultimate testing and validation in cells and tissues.\nWe firmly believe that utilizing micromolar and not millimolar concentrations of 2t16 more closely resembles cellular contexts – there is no argument here as nanomolar levels in cells are reported in the literature, yet levels can be amplified to micromolar upon inhibition of the S1P lyase. The experiment with 40,000-fold 2t16 is to saturate the binding curve and demonstrate that the effect does NOT happen despite the incredible molar excess, which is both a rational and anticipated investigation. This singular technical approach does not undermine the study as suggested by the Reviewer’s distortion of the methodologies. While the Reviewer appears to make the assertion that the specifics of in vitro model systems are unimportant because they are never truly physiological, we wish to remind them that the role of substrate concentration is extremely relevant to a proposed covalent reaction and thus warrants highlighting. To ensure our intentions are clear in the manuscript, we adjusted the text accordingly.\n7. Lingering structural methodology concerns.\nThe manuscript remains heavily reliant on static docking calculations, which constitute a minimal and low-rigor approach given the central role of computation in their conclusions. While the authors argue that molecular dynamics (MD) simulations are beyond scope, this is unpersuasive; MD is precisely the method needed to capture conformational variability, address structural perturbations (e.g., α8 deletion), and evaluate stability of binding modes. The current strategy does not adequately represent the relevant ensembles or intermediates, and the terminology conflating docking with simulation is misleading. At minimum, the authors should employ explicit solvent MD sampling of existing structures, relax mutant models, and dock to representative ensembles. Without this, the work does not meet basic standards for a high-impact publication.\nWe certainly appreciate the suggestion to enhance our manuscript with additional in silico data. Yet, molecular dynamics simulations are inherently theoretical and rely on force fields that approximate molecular behavior, and without experimental validation (e.g., NMR), the results remain purely speculative. For our approach, we instead performed NMR as the first step prior to rigid-docking and the identification of BAX mutants to investigate in the manuscript. We realize this may be perceived as a departure from the normal workflow, but as early conformational changes of monomeric BAX beyond BIM stimulation are not well characterized at the structural level, we focused on the structure of quasi-active BAX bound by BIM-SAHB with α8 removed (PDB 2K7W), and supported these observations to include the inactive monomer structure (PDB 1F16) to explore BAF topography in both inactive and triggered monomeric BAX.\nIn good faith, and to ensure our strategy was acceptable, we recently discussed this Reviewer’s molecular dynamics comment with several experts in the field, and all concluded that given our workflow to first perform NMR and then subsequent experimentation with several mutants to support the NMR and rigid docking, the addition of molecular dynamics would not influence the conclusions of our paper, yet it would require months of effort with parameters that are largely speculative and largely assumed based on biochemical systems rather than cell biology. If we were asserting specific binding affinities, then advanced MD simulations would likely provide more precise metrics; however, our docking-based comparisons are relative in nature (e.g., a modification results in disordered binding predictions), are used as hypothesis-generating, and are experimentally validated with structural mutants. As such, the Reviewer's suggestion would not change the following conclusions:\n1. The BAF is present in all ensembles of the structures modeled in this work (lines 285–287, 299–302, Figures S4F–G);\n2. The BAF cavity is represented in several BAX structures, including crystal structures and solution NMR structures (lines 574–579, Figure S10D);\n3. Mutagenesis of BAF-forming residues selectively loses sensitivity to 2t16 while remaining sensitive to BIM activation (lines 416–424; Figures 6G–H, S8C–F).\nGiven the utilization of more than 15 techniques and analytical tools, integration of numerous cell biology concepts, and the description of a new domain with the BCL‑2 family, we do not agree with the specious comment that our applied expertise within the manuscript fails to meet basic standards for a high-impact publication.\nTo summarize, the authors work so hard at justifying the new model that they undermine their credibility by making comparisons and justifications that aren’t objectively effective or necessary based on the data provided. Making the suggested textual refinements, with an overall more objective approach to data interpretation would allow the manuscript to stand as a more constructive extension of previous models rather than a competing reinterpretation, whilst fully crediting established physiological data and situating the proposed mechanism within the known biochemistry of lipid electrophiles. It may be \"OK\" to directly say, this is what we speculate based on our results despite the caveats, some of our own data (and prior data) are inconsistent with our new model, and even quite inconsistent with what the reader might expect from lipid electrophiles and how they are used in the study. At the end of the day, a more circumspect handling seems not only justified, but could ultimately come across more genuine and compelling.\nWe appreciate the reviewer’s expert and candid advice, and we believe that the integration of these comments has improved the accuracy, tone, and data interpretation. Thank you."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors’ responses address several points, but many rely more on rhetorical framing than on directly engaging the underlying scientific issues. To be constructive and avoid unnecessary back-and-forth, I offer a few specific examples where clarification or correction would strengthen the manuscript. My intention is sincerely to help refine the discourse to ensure that the authors’ interpretations align with biochemical principles and the data presented.\nWe thank the Reviewer for their constructive criticism and suggestions for how we may further clarify and strengthen the manuscript. We interpret that several of the Reviewer's comments regarding our word choices were perceived as weakening published works, but our intentions were to aid readers in following our experimental progression. In good faith, we have made additional text adjustments to help avoid any misinterpretations of our language and address several of the points raised by the Reviewer\nLine 4, Title: Consider whether “lipids” should be singular, as the study focuses on one lipid species.\nWe understand the Reviewer's comment. However, we demonstrate (using in silico and experimental data) that similar long-chain alkenals also activate BAX, thus leading to our conclusion that the alkenal length is a determinant of the interaction (likely due to increased specificity for the BAF) (Figures 5, S5, S6, and S7). While hexadecenal is likely the predominant bioactive lipid in the cell, other long-chain aldehydes may similarly interact with BAX. As such, we believe the current title is accurate to the findings and keeps the field open to additional interactions and discovery.\nLines 34–35, Abstract: The phrase “mechanisms remain underexplored” is not strictly accurate, as prior work included detailed structure–function studies. The sentence reads clearly without this clause.\nWe have removed the phrase as suggested.\nLine 84, Introduction: The phrase “requirement for 2t-hexadecenal in BAX pore formation” mischaracterizes earlier covalent studies, which examined BAX monomer activation rather than strictly pore formation.\nThe Reviewer has misunderstood this sentence. The quoted phrase refers to the original Cell 2012 paper, which primarily assessed BAX activation by pore formation, not the covalent studies; the second half of the sentence describes the recent covalent studies. Nevertheless, we have changed the phrase to \"the requirement for 2t‑hexadecenal in BAX-mediated MOMP…\" to avoid this misunderstanding.\nLines 106–107, Results: The external-treatment approach remains difficult to interpret given the compound’s high intrinsic reactivity. It is unknown whether sufficient cytosolic levels can be reached to achieve a non-covalent interaction with BAX, as opposed to non-specific induction of stress. The term “validated ectopic hexadecenal” is not supported experimentally.\nThe term \"validated ectopic hexadecenal\" refers to the control mass spectrometry experiment in which we validate the molecular weight of our reagent, as previously requested in the review process (Figure S1A). There is no suggestion that \"validated\" refers to the results of the ectopic treatment experiment. To avoid this confusion, we have removed the word \"validated\".\nThe Reviewer maintains that we overstate the conclusions of these assays and we politely do not agree. We previously addressed this comment and remind the Reviewer that we explicitly state the limitations of this approach and its conclusions in the text. Moreover, these limitations are the rationale for directly investigating the effect of hexadecenal on BAX using model membrane systems and several other experimental approaches using BAX protein. We believe that our approaches (including limitations), interpretations, and reasoning are transparently delivered to the reader and that our conclusions evolve with each experiment over the course of the paper.\nLines 130–131, Results: The statement that the authors use LUVs “without altering the biochemistry of the LUV membrane” doesn’t really apply; LUVs are synthetic systems defined entirely by their constructed compositions.\nThe lipid species and ratios used to make LUVs are widely standardized in the apoptosis field as they were specifically selected to mimic the major lipids comprising the outer mitochondrial membrane (as published in Kuwana et al., 2002). Our statement that we did not deviate from the standard composition is specifically to distinguish our approach from prior works (in which hexadecenal was incorporated into the LUV formulation) and to make it clear to the reader that we are not merely replicating prior investigations. We believe this is an important distinction especially when our conclusions propose an alternative mechanism to what has previously been published. We have added the appropriate reference to contextualize our statement and the importance of LUV composition.\nLines 142–144, Results: This is a key area needing clarification. Both control compounds—hexadecanal (double bond reduced) and the hydrazone (aldehyde eliminated)—lack electrophilicity because the distinctly reactive α,β-unsaturated aldehyde moiety is disrupted in each case. Although the hydrazone species still contains a double bond, it is not “reactive” (electrophilic) without conjugation to the aldehyde. Readers will recognize that both controls remove Michael acceptor capacity. Thus, these negative results are much more consistent with loss of covalent reactivity than with selective loss of a non-covalent interaction that happens to require both of these molecular features. This is important because both compounds are treated as specificity controls across multiple assays (thermophoresis [lines 158-159], FLAMBE [lines 216-217], SEC [lines 247-248], results paragraph introduction [lines 335-336], etc.), so accurate interpretation here is especially important.\nThe Reviewer’s interpretation of these experiments and controls has evolved over the review process. The decision to use a hydrazone derived molecule was in response to this Reviewer, who had requested a control molecule \"which maintains the double bond but removes the electrophilic aldehyde functionality\". This was specifically accomplished by the 2t16‑NN‑EtOH hydrazone molecule; the Reviewer has now changed their interpretation, thus undermining their original concern we addressed.\nIn this third set of comments, the Reviewer suggests that the ability for Michael reaction requires both the aldehyde moiety and a proximal double bond, and therefore the 2t16‑NN‑EtOH molecule may not exhibit the same reactivity despite conservation of the double bond. This is not our area of expertise, and we cannot comment on the validity of this point. Therefore, in good faith, we added a comment discussing this possibility and that the loss-of-function of the two molecules could suggest a chemical reaction mechanism.\nWe remind the Reviewer that the limitation of these molecules and their conclusions is precisely why we then study a cysteine-deficient BAX mutant to demonstrate that our observed phenotype is not due to covalent modification. This critical control is what clarifies the interpretation of the modified molecules and corroborates the existence of a non-covalent interaction. In our opinion, we present no misinterpretation in subsequent assays as suggested by the Reviewer – the use of these molecules (and the cysteine-deficient mutant) is to demonstrate conservation of the phenotype between assays. In service of this goal, we have moved the 2t16 binding curve into the main figures (now Figure 2B) and put the comparison of 2t16 and hexadecanal in the supplement (now Figure S2B).\nAs a separate consideration, we point out that the original work studying the role of hexadecenal in BAX activation utilized cis-11-hexadecenal. While the Reviewer now suggests that the double bond must be proximal to the aldehyde moiety, it is worth noting that this is not true in cis‑11‑hexadecenal, yet the aldehyde activated BAX regardless. This would suggest: 1) that the original work was in fact studying the non-covalent interaction, thus supporting our conclusions; and/or 2) that the Reviewer's supposition regarding the origin of the electrophilicity is not accurate. We chose not to explore this point within the work as it was a divergence from the main biology and instead we focused on the structure-function relationship of BAX utilizing experimental and computational approaches corroborated with numerous rationally-designed loss-of-function mutations.\nLine 196, Results: The distinction made regarding “direct treatment” is not meaningful, as prior studies also used direct treatment models.\nIn this section of the Results, we posit that it is important to remind the reader that we utilized a model in which BAX encounters the aldehyde in solution prior to membranes (i.e., when the BAX conformer resembles the structures used later in the work – namely, 1F16 and 2K7W). The sentence goes on to state that membrane incorporation methods would have hexadecenal interacting with the conformers that BAX adopts at the membrane, which include multimeric and oligomeric structures. These conformers are vastly different, and it is important for our readership to be reminded which part of the BAX activation continuum we are investigating with each experimental approach. Furthermore, this point is provided to explain our progression into a different BAX activation model system – namely, one that observes BAX activation in solution prior to membrane interactions.\nLines 254–255, Results: A concluding statement about lipid-BAX association at a specific stage of the activation pathway is made without evidence of the interaction in a cellular or cell lysate context.\nThe sentence in question reads: \"These data collectively demonstrate that hexadecenal promotes monomeric BAX activation downstream of BCL‑2 protein interactions, following activation by direct activators, and before interactions with the OMM.\" The data at this point demonstrate a requirement for BIM-triggering and increased multimerization in solution prior to interactions with a membrane (Figure 4). Therefore, the referenced sentence accurately summarizes the data and conclusions at that point in the paper. There is no mention or suggestion of the larger cellular context because, as the Reviewer points out, those were not experimentally examined. This does not undermine the data or conclusions as the underlying biology of the BCL‑2 family was discovered, and continues to be studied, using biochemical approaches, and our interpretation is supported by the scientific literature and current understanding of how the BCL‑2 family functions. However, to avoid the possibility of inaccuracy, we changed \"the OMM\" to \"a membrane\" in case this was the focus of the Reviewer's concern.\nLines 354–355, 402–404, 433–435, Results: The authors appropriately acknowledge the limitations of rigid docking, including artifact risk and inability to determine ligand orientation. However, given these limitations, it is premature to draw a declarative conclusion that the aldehyde “resides in the depth of the BAF” without more informative structural or computational approaches. Incorporating even basic molecular dynamics simulations is widely considered an industry standard for such claims in a high-impact journal.\nThe Reviewer raised this comment twice before and we provided a detailed and thorough response as well as incorporating the requested modifications into the text (as noted above and in the previous two Reviewer’s Response documents).\nTo briefly reiterate our position on this matter:\n1. Molecular dynamics is fundamentally a theoretical computational approach, and instead we provide experimental validation through a variety of techniques and rational design of specific and selective loss-of-function mutations. Any additional insights gained by MD would be entirely speculative in nature.\n2. Prior, this Reviewer suggested MD for solvent sampling, model relaxation, and then docking to a representative ensemble. Critically, this would only potentially alter the BAX model, and the docking of hexadecenal would then be subsequently conducted using the same method as the current data – the MD simulation does not perform or replace the docking efforts. Given that the BAX models utilized in this work were selected from solution NMR ensembles, it is unclear how docking against a single \"representative ensemble\" would be fundamentally different than the deposited structures that were experimentally determined.\n3. Fundamentally, the addition of MD as previously suggested by this Reviewer would not change the following conclusions: i) the BAF is present in all ensembles of the structures modeled in this work (Figures S4F–G); ii) the BAF cavity is represented in several BAX structures, including crystal and solution NMR structures (Figure S10D); iii) studies mutating BAF-forming residues demonstrated a selective loss-of-function for hexadecenal while remaining sensitive to BIM activation (Figures 6G–H, S8C–F).\nGiven the utilization of more than 15 techniques, approaches, and analytical tools, as well as the description of a new domain within the BCL‑2 family, we do not agree with the specious comment that our applied expertise within the manuscript fails to meet basic standards for a high impact journal.\nLines 500–501 and 522–523, Discussion: The suggestion that prior results may reflect “the use of millimolar concentrations of 2t-hexadecenal”, coupled with the assertion that BAX is “unlikely to experience millimolar concentrations…during activation in the cytosol,” is misleading. Earlier studies also employed micromolar concentrations as well as endogenous, physiologic levels present in mitochondrial membranes. It is worth noting that covalent mechanisms generally require far lower effective concentrations than non-covalent interactions to exert biological effects in a cellular context, so the focus on millimolar levels does not meaningfully address the underlying issue.\nThe Reviewer has provided their opinion on this matter previously, and we reiterate that our discussion on the topic is valid and warranted when attempting to rationalize how each of the manuscripts – both prior and our own – result in divergent conclusions while not being mutually exclusive. While this Reviewer holds this opinion, it is worth noting that another reviewer (Reviewer 2) explicitly requested commentary on the difference in approaches and how it may have resulted in different findings. We believe that the Discussion is the appropriate place for these types of considerations as it is how we can best position our findings in the context of prior works, while supporting the conclusion that the different results are without conflict.\nThe assertion that covalent interactions require \"far lower effective concentrations than non-covalent interactions to exert biological effects in a cellular context\" is a mischaracterization of the point we raise in the text. In a cellular context, there are several factors that may facilitate covalent interactions (for example, enrichment and sidechain positioning at membranes), and this is precisely the point we make – that the structure of activated, membrane-associated BAX structures have exposed cysteines for targeting and likely sit at sites enriched in hexadecenal (Figure S10). By comparison, the prior work demonstrated that inactive cytosolic BAX was not modified by hexadecenal at micromolar concentrations, whereas millimolar concentrations instigated the covalent reaction. There is no disagreement here, as the point in question compares the results from experiments studying inactive, monomeric BAX in solution; we go on to posit that membrane-associated BAX would be more primed for modification, thus aligning with the approaches and results of the prior work. Additionally, we would like to point out that in both examples that the Reviewer cites, we explicitly state that we are discussing BAX in solution/cytosol.\nFinally, we believe that our position is clear – in both the manuscript text and in multiple responses to the Reviewer's comments – we do not dispute the previous findings and offer consideration and key methodological differences to rationalize how each manuscript identified distinct (and non-conflicting) conclusions regarding BAX biology. We edited the text to avoid reasonable misunderstanding or misinterpretation of our logic, approach, data, or conclusions. Finally, we acknowledge that the manuscript has been improved during the revision process as we have refined our discussion and interpretations at multiple steps to appease this Reviewer's concerns."}, "author_response": ""}]

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