1. Volume 27, Issue 2, Pages 359-373.e6 (April 2019)
BAX Activation: Mutations Near Its Proposed Non-canonical BH3 Binding Site Reveal Allosteric Changes Controlling Mitochondrial Association 
Michael A. Dengler, Adeline Y. Robin, Leonie Gibson, Mark X. Li, Jarrod J. Sandow, Sweta Iyer, Andrew I. Webb, Dana Westphal, Grant Dewson, Jerry M. Adams 
Cell Reports 
Volume 27, Issue 2, Pages e6 (April 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 359-373.e6DOI: (10.1016/j.celrep.2019.03.040)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
BAX Translocation and MOM Integration Can Be Mediated Independent of Groove Binding
(A) BAX-mediated cytochrome c release from Bak−/− MLM shows that K21E retains full WT activity, whereas two groove mutants are inert. MLM were supplemented with 10 nM recombinant BAX and graded tBID (1 h, 37°C). After fractionation into supernatant and pellet, immunoblotting assessed cytochrome c release.
(B) Like WT and K21E BAX, groove mutants G108V and R109D still translocate to the MOM and integrate upon tBID treatment. Peripherally attached and membrane-integrated BAX were determined by carbonate extraction on BAX-supplemented Bak−/− MLMs treated with tBID as above, and peripheral and integrated fractions were immunoblotted for BAX.
(C) Whereas WT BAX and K21E oligomerize, the groove mutants translocate to the MOM but stay monomeric. Bak−/− MLMs supplemented with 50 nM BAX were treated with 10 nM tBID (2 h, 37°C), and membrane fractions solubilized with 1% digitonin were run on BN-PAGE and immunoblotted.
(D) BAX sub-populations distinguished by limited PK proteolysis. In inactive WT BAX, the major cleavage is after Phe176 in α9, whereas activated BAX is cleaved instead in the α1-α2 loop, mainly after Met38 (Bleicken and Zeth, 2009; Goping et al., 1998; Robin et al., 2018).
(E) Upon a death stimulus, unlike the active conformation assumed by WT BAX and K21E, the groove mutants translocate to the MOM but retain an inactive conformation. Bax/Bak DKO MEFs expressing the BAX variants were pre-incubated with caspase inhibitor Q-VD.oph (25 μM) for 1 h, then treated with 5 μM etoposide. After 16 h, the cells were permeabilized with 0.025% digitonin and treated with PK (20 min on ice) before fractionation, carbonate extraction, and immunoblotting for BAX.
(F) Unlike WT and K21E BAX, the groove mutants do not expose the N-terminal 6A7 epitope upon a death stimulus. DKO MEFs expressing the human BAX variants, treated as in (E), were solubilized in 1% (3-((3-cholamidopropyl) dimethylammonio)-1 (CHAPS), and activated BAX was immunoprecipitated with antibody 6A7. All immunoblots are representative of at least two independent experiments.
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4. Figure 2
An Activator BH3-Only Protein Can Be Disulphide Linked near the Proposed BAX Rear Site
(A) Model of BIM SAHB bound to the BAX rear site (PDB: 2K7W) (Gavathiotis et al., 2008). The BIM SAHB peptide (red) lies across BAX helices α1 (blue) and α6 (green) in the BAX rear site, placing BAX E146 at the end of α6 near BIM E145 at the N terminus of the BH3 peptide.
(B) BH3 peptides readily cross-link to BAX E146C in α6. BAX L45C/M137C/E146C, with L45C tethered to M137C (Figure S2A) using 0.5 mM GSSG (5 h, 35°C), was purified on gel filtration (GF) (Figure S2B). The tethered monomer (50 μM) was incubated with BID BH3 I83C or BIM BH3 E145C (1 h, room temperature [RT]) and the peptide coupled with cross-linker BMOE or oxidant GSSG (30 min, RT). Samples were run on reducing (BMOE samples) or non-reducing (GSSG samples) SDS-PAGE and gels stained with Coomassie blue. Arrowhead, peptide-linked BAX; ∗, residual tethered BAX.
(C) BID BH3 peptides can also efficiently link to BAX E146C bearing mutations P168G/W170A, which keep α9 in the groove. The BAX variant (50 μM) was incubated with BID I83C peptide and GSSG or BMOE to induce linkage (1 h, RT) and samples run on non-reducing SDS-PAGE.
(D) Top: the cysteine replacements in BAX P168G/W170A and full-length tBID screened for linkage (PDB: 2K7W). Bottom: aligned BH3 domains of BIM, BID WT, BID I83C, BID M97C, and BID I101C, with conserved hydrophobic BH3 residues (h1–h4) highlighted.
(E) Tests of full-length tBID-hemagglutinin (HA) cross-linking to BAX P168G/W170A with the cysteine substitutions in (D). BAX variants (200 nM) were incubated with tBID-I83C-HA, tBID-M97C-HA, or tBID-I101C-HA (400 nM, 15 min, ice) or no tBID, disulphide linkage was induced with 1 mM CuPhe (15 min, ice), and BAX was immunoprecipitated with an antibody (3C10) that recognizes both active and inactive BAX or HA (tBID). Samples run under non-reducing conditions were immunoblotted for BAX or HA (for tBID). Inputs are shown in Figure S2C. Arrows, tBID:BAX heterodimers; #, unspecific band.
(F) Cys null tBID reduces linkage to BAX P168G/W170A cysteine variants. BAX variants (200 nM) were incubated with equimolar (1×) or threefold molar excess (3×) of tBID-WT(Δcys)-HA for 15 min on ice before incubation with tBID-I83C-HA or tBID-M97C-HA (400 nM, 15 min, ice) and induction of disulphide linkage and immunoprecipitation as in (E). Inputs are shown in Figure S2D. Arrows, tBID:BAX heterodimers. All gels and immunoblots are representative of at least two independent experiments.
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5. Figure 3
Obstructive Labeling of BAX Helices α1 and α6 Attenuates BAX Activation
(A) BAX (PDB: 1F16) showing the cysteine replacements along BAX α1 (blue) and α6 (green).
(B) PEG-maleimide efficiently labels all the replacements. BAX variants (50 μM), labeled or not with PEG-maleimide (0.5 mM, 30 min, RT), were run on SDS-PAGE and stained with Coomassie.
(C and D) Impact of cysteine substitutions and PEG-maleimide labeling on BAX-mediated cytochrome c release. Bak−/− MLMs were supplemented with 10 nM of unlabeled or PEG-maleimide-labeled recombinant BAX and graded tBID (1 h, 37°C). Supernatant and pellet samples were immunoblotted for cytochrome c (see Figure S3) and percent cytochrome c release determined densitometrically. Data are means ± SEM of at least three independent experiments.
(C) Unlabeled cysteine substitutions did not affect BAX activity.
(D) PEG-maleimide labeling of certain α1 or α6 residues strongly attenuated BAX-mediated cytochrome c release.
(E) PEG-labeling reduces 6A7 exposure on BAX A24C and blocks it on R134C (compare filled and unfilled arrows on 6A7 immunoprecipitates). Mitochondrial samples, solubilized in CHAPS, were immunoprecipitated with 6A7 antibody. ∗, immunoglobulin G (IgG) heavy chain. PEG-labeled BAX runs more slowly (open arrows). As expected, no BAX with 6A7 exposed appeared in cytosolic (supernatant) fractions (Figure S3C).
(F) BAX-mediated cytochrome c release from Bak−/− MLM shows that BAX mutants A24D, G138D, and A24D/G138D retain full WT activity. MLM were supplemented with 10 nM of recombinant BAX and graded tBID (1 h, 37°C). After fractionation into supernatant and pellet, immunoblotting was assessed cytochrome c release. The gel in (B) and immunoblots in (E and F) are representative of at least two experiments.
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6. Figure 4 Mutations in BAX α1 and α6 Impair BAX Apoptotic Activity
(A) BAX triple-alanine mutagenesis scan of α1 and α6. In α1, the key conserved BH4 residues are marked.
(B and C) Mutagenesis of certain α1 and α6 residues strongly impairs BAX function, as shown by representative blots in Figure S4.
(B) The mutants were analyzed as in Figures 3C and 3D. Black lines are WT and red the mutant.
(C) The nine single-alanine variants of triplets H1f, H6b, and H6d, analyzed as above and in Figure S4C. Data in (B) and (C) are means ± SEM of at least three independent experiments.
(D) Model of BAX (PDB: 2K7W) (Gavathiotis et al., 2008) showing that the residues in the central areas of α1 or α6 identified by single alanine mutants (red) are very close to residues (blue) on which obstructive labeling most strongly impaired BAX function (from Figure 3D).
(E) The single-alanine mutations block N-terminal 6A7 epitope exposure upon tBID treatment. Bak−/− MLMs treated with 10 nM of recombinant BAX and graded tBID (1 h, 37°C) were solubilized in 1% CHAPS and immunoprecipitated with 6A7 antibody.
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7. Figure 5
BAX α1 and α6 Residues Are Important for BAX Translocation and MOM Integration
(A) Tests of activity of BAX α1 and α6 mutants in cells. Bax/Bak DKO MEFs expressing the indicated BAX variants were treated, or not, with 5 μM etoposide for 12 or 24 h and mitochondrial membrane potential determined by tetramethylrhodamine, ethyl ester (TMRE) staining and fluorescence-activated cell sorting (FACS) analysis. Triple- and cognate single-alanine mutants have the same color. Data are means ± SEM of at least three independent experiments. Statistical analysis of differences from WT BAX was by two-way ANOVA (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < ). Insert, expression level of the BAX variants assessed by immunoblotting, with HSP70 as a loading control.
(B) BAX α6 mutations block 6A7 epitope exposure upon etoposide treatment. MEFs expressing the BAX variants were pre-incubated with 25 μM Q-VD.oph for 1 h and then treated with 5 μM etoposide. After 16 h, cells were solubilized in 1% CHAPS prior to immunoprecipitation with 6A7 antibody.
(C) Mutations in α1 and α6 reduce MOM translocation and integration of BAX. Bax/Bak DKO MEFs expressing the α1 and α6 variants were pre-incubated with 25 μM Q-VD.oph for 1 h, treated with 5 μM etoposide for 16 h, subjected to carbonate extraction and fractions run on SDS-PAGE, and immunoblotted for BAX. (B and C) show representative blots.
(D and E) Densitometric analysis of carbonate extracts (as in C) indicates that α1 and α6 mutations affect BAX translocation and/or integration at different stages.
(D) At steady state, several mutants show reduced peripherally attached BAX (top) and/or membrane-integrated BAX (bottom), as percent of total BAX.
(E) After etoposide treatment, most mutants show less total membrane-integrated BAX than WT BAX (top). However, when the proportion of integrated BAX is plotted as fold increase over steady state level (bottom), only three α6 mutants show a marked reduction in integrated BAX: H6b, H6d and I133A. Significantly, these are the mutants with the greatest reduction in BAX killing (A).
Data in (D) and (E) are means ± SEM of four independent experiments. Significant differences from WT BAX were revealed by two-tailed paired t tests (∗p < 0.05; ∗∗p < 0.01) or a one-tailed paired t test (#p < 0.05).
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8. Figure 6
BAX α1 and α6 Mutations Reduce Cross-Linkage to tBID and Alter BAX Conformation
(A) Mutations in α1 and α6 reduce disulphide linkage of tBID to BAX α6. Recombinant BAX E146C/P168G/W170A with the indicated mutations (200 nM) was incubated with tBID-I83C-HA or tBID-M97C-HA (400 nM, 15 min on ice) and disulphide linkage induced with 1 mM CuPhe (10 min on ice). Samples were immunoprecipitated with BAX 3C10 or HA (for tBID), run under non-reducing conditions, and immunoblotted for HA (tBID) or BAX. Arrows indicate tBID:BAX heterodimers.
(B) Mutations in α1 and α6 alter the conformation of the α1-α2 loop and α9 exposure, as does the α8-α9 loop mutant P168G. BAX α1 and α6 variants and BAX P168G (1 μM recombinant protein) were treated with PK (20 min on ice), run on SDS-PAGE, and immunoblotted for BAX. Cleavage in the α1-α2 loop and α9 is illustrated by PK-cleaved samples from Bak−/− MLMs supplemented with BAX WT ± tBID (lanes 1–3). The immunoblots in (A) and (B) are representative of at least two independent experiments.
(C) The inferred conformers of the α1 and α6 mutants and P168G.
(D) Crystal structure of BAX W139A reveals allosteric conformation changes. The W139A structure (light orange) (PDB ID: 6EB6) shows the helical bundle fold typical of non-activated BAX, with helix α9 in the groove formed by helices α3, α4, and α5 (see also Figure S6B). The overlay of its structure with those of BAX P168G (blue, PDB ID: 5W60) and mouse WT BAX structure (mmBAX, dark red, PDB ID: 5W61) shows their conserved overall protein fold (middle view). The left insert shows that the W139A mutation causes a slight collapse of α4 toward 139 on α6, with the R89 sidechain moving into the space that the large tryptophan occupied; however, the relative backbone positions of helices α4 and α6 are unchanged beyond positions 93 and 139, respectively, as shown by the conserved positions of F100 in α4 and F143 in α6. The right insert shows that the rotamers of F105 in the α4-α5 loop and W151 in α7 of W139A differ from those in WT BAX but that W151 of W139A aligns closely with W151 in P168G, indicating that the W139A mutation has induced allosteric changes in the α4-α5 loop and α7.
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9. Figure 7
A BAX Activation Model and the Inferred Impact of α1 and α6 Mutations on BAX Activation
(A) Model for BAX activation by sequential BH3 binding to two sites. Upon a death stimulus, BH3-only proteins, such as BID or BIM, engage the BAX α1 and α6 site and induce conformation changes that promote release of α9 to increase BAX translocation to and integration into the MOM. This facilitates BH3 binding to the BAX canonical groove, which elicits exposure of the BAX N terminus and BH3 domain, latch (α6–α8) dissociation from core (α2–α5), BAX BH3-in-groove dimerization and oligomeric pore formation.
(B) Multiple BAX conformers at steady state. WT BAX appears to assume multiple conformations, including cytosolic monomers with α9 exposed or tucked into its canonical groove and monomers peripherally associated with the MOM or integrated within it, including some associated with VDAC2; the MOM-associated or integrated BAX can retro-transpose to the cytosol. Mutations in α1 and α6 can cause cytosolic BAX to assume conformations less able to engage the MOM, due to exposure of the α1-α2 loop and resulting sequestration of α9 in the groove (see Discussion).
(C) The shift in BAX conformers in response to a death stimulus. BH3 engagement of the α1 and α6 site stabilizes WT BAX in a conformation with α9 exposed and, therefore, favors increased BAX MOM association and integration. The α1 and α6 mutations reduce BH3 binding to the site and change the BAX conformation, allosterically impeding α9 release. Whether these conformation changes occur in the cytosol or on the MOM (or both) is not known.
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