1. Volume 63, Issue 3, Pages 485-497 (August 2016)
An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway 
Thomas P. Garner, Denis E. Reyna, Amit Priyadarshi, Hui-Chen Chen, Sheng Li, Yang Wu, Yogesh Tengarai Ganesan, Vladimir N. Malashkevich, Emily H. Cheng, Evripidis Gavathiotis 
Molecular Cell 
Volume 63, Issue 3, Pages (August 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 63, 485-497DOI: (10.1016/j.molcel.2016.06.010)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 BAX Forms an Inactive Dimer Conformation
(A) Recombinant BAX (rec. BAX) and cytosolic extracts from WT MEF, OCI-AML3, A375, DKO MEF reconstituted with human BAX, and WT MEF treated with 1% Triton X-100 (cyt. BAX + 1% Triton) were analyzed by Superdex 200 (HR 10/30) gel filtration and anti-BAX immunoblot. The centers of the eluted monomer (M) and dimer (D) peaks are indicated.
(B) Anti-apoptotic BCL-2, BCL-XL, MCL-1, and BAX protein levels in MEFs determined by western blot analysis of separated cytosolic and mitochondrial fractions as confirmed by the anti β-tubulin and anti-VDAC immunoblots, respectively.
(C) SEC fractions of cytosolic extracts of MEFs containing dimeric BAX WT (fractions 13.5 and 14.5 mL) failed to be immunoprecipitated with the 6A7 antibody. When the fractions corresponding to dimers were incubated with 1% octylglucoside detergent, the 6A7 epitope was then exposed on BAX, and BAX could be immunoprecipitated with the 6A7 antibody.
(D) Dimerization of purified monomeric recombinant BAX at the indicated concentrations or prior to concentration (pre-concentration) was analyzed by Superdex 75 (HR 10/30) gel filtration. The peaks of monomer (M) and dimer (D) were eluted at ∼11.8 mL and ∼10.4 mL, respectively. Total amount of BAX was kept constant in different samples.
(E) Representative dynamic light scattering data of BAX monomers and dimers taken from the SEC elution peaks in (D).
(F) Dimerization of purified monomeric BAX 4M mutant at pre- and post-concentration to 10 mg/mL was analyzed by Superdex 75 (HR 10/30) gel filtration.
(G) Quantification of BAX dimerization formed by BAX WT and BAX 4M based on SEC peaks at 10 mg/mL.
(H) ANTS/DPX-encapsulated liposomes were incubated with SEC-isolated BAX monomers or dimers without or with BIM SAHBA at the indicated concentrations. The release of entrapped fluorophore monitored with time is shown.
(I) Liposome release assays as in (H), with each bar indicating the total liposomal release at 90 min.
Data shown in (A)–(G) are representative of three independent experiments with similar results. Data in (H) and (I) are mean ± SD for assays performed in triplicate and two independent experiments. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2 Crystal Structure of the Inactive BAX Dimer
(A) Ribbon representation of the BAX dimer crystal structure. Helices (α) and loops (L) are depicted on the structures. Dimerization interaction interfaces are shown in pink and purple for each BAX protomer. Dotted line represents the missing α1-α2 loop residues that have weak electron density. Secondary structure cartoon representation of BAX, highlighting the location of the BCL-2 homology domains (BH) in light green, the transmembrane region (TM) in dark green, and the dimerization interaction interfaces in pink and light blue, is shown above the crystal structure depiction.
(B) Ribbon representation of each BAX protomer in view related to the representation in (A) at 90° rotation around a vertical axis, showing the N-terminal and C-terminal dimerization interfaces.
(C) Structural alignment of the solution structure of BAX (green) (PDB: 1F16) and BAX protomer A (gray) within the BAX dimer. Views centered on helix α5 (top view) and the N-terminal activation site (right).
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3 Determinants and Mechanism of Inactive BAX Dimerization
(A) Calculated vacuum electrostatics of the C-terminal and N-terminal dimerization interfaces from each BAX protomer showing the position of complementary hydrophobic, polar, and charged residues.
(B–E) Cartoon representation of the BAX dimer showing the interacting protomers in pink (C-terminal dimer interface) and violet (N-terminal dimer interface). The interacting residues are shown in sticks and are highlighted in three different regions of the dimerization interface: (C) hydrophobic core, (D) and (E) hydrogen bonds and salt bridges of the dimerization interface.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4 Structural Insights Suggest a BAX Autoinhibition Mechanism
(A) Structural comparison of the BAX:α9 interaction from the BAX dimer structure and BAX:BIM BH3 interaction (PDB: 2K7W).
(B) Ribbon representation of the BAX:α9 interaction showing α1 residues, A24, L25, Q28, and Q32, interacting with residues Q52, V50, and D48 of the α1-α2 loop of the same BAX molecule (purple) and with the adjacent BAX molecule through α9 residues I175 and F176 and α4-α5 loop residues R109 and N106 (pink).
(C) Surface representation of the BAX:α9 interaction showing residues of the N-terminal activation site as hydrophobic (yellow), positively charged (blue), and negatively charged (red) residues and BAX α9 helix hydrophobic residues in yellow sticks.
(D) Ribbon representation of the BAX:BH3 interaction showing α1 residues, A24, L25, and L27 forming interactions with conserved hydrophobic residues F159 and I155 of BIM BH3. Residues Q28 and Q32 of BAX interact with residue N160 of BIM BH3.
(E) Surface representation of the BAX:BH3 interaction shows that BIM BH3 helix (cyan) forms hydrophobic and electrostatic interactions with residues of α1 and α6.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 5
The Autoinhibited Dimeric Form of BAX Dissociates to BAX Monomers before Its Activation
(A) Dimerization of purified monomeric recombinant BAX WT and mutants of the N-terminal interaction surface (purple bars) and the C-terminal interaction surface (pink bars) were analyzed by SEC and quantified by integration of areas under observed monomer and dimer peaks. Residues mutated are shown on the ribbon structures of BAX.
(B) Cytosolic fractions purified from DKO MEFs reconstituted with BAX WT or mutants were analyzed by SEC using Superdex 200 (HR 10/30) and anti-BAX immunoblot.
(C) Representation of the relationship between the inactive BAX dimers, BAX monomers, and the BAX oligomers.
(D) Inactive BAX 4M dimers under oxidizing conditions dissociate into monomers in response to BIM SAHBA. Reduction of the internally crosslinked BAX 4M mutant dimer using 20 μM DTT followed by incubation with BIM SAHBA dissociate dimers into monomers and induce the oligomerization of BAX.
(E) Quantification of percentage BAX monomers and percentage BAX oligomers based on SEC peaks shown in (D) and Figure S5C. Data are representative of three independent experiments with similar results.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 6
Inactive BAX Dimer Regulates BAX Activation and BAX-Mediated Apoptosis
(A) Viability assays of DKO MEFs transiently transduced with retrovirus expressing human BAX WT or mutants as measured by annexin-V staining. P < 0.05 for BAX mutants compared with BAX WT.
(B) Caspase-3/7 activation assays of DKO MEFs stably transduced with human BAX WT or mutants as measured by caspase-3/7 activation assays upon STS treatment at 0.5 or 1 μM for 12 hr.
(C) Caspase-3/7 activation assays of DKO MEFs stably transduced with human BAX WT or mutants upon 1 μM STS treatment at 3, 6, and 12 hr.
(D) BAX WT and BAX P168G cells were treated with 1 μM STS for 6 hr; BAX E75K cells were treated for 3 hr because of faster cell death. Cytosolic and mitochondrial fractions were separated and analyzed by SEC and anti-BAX immunoblot. Data shown in (A), (B), and (C) are mean ± SD from three independent experiments and in (D) are representative of three independent experiments with similar results.
(E) Autoinhibited dimeric BAX regulates the BAX activation pathway. Activation of the cytosolic BAX monomers (1) is initiated by BIM BH3 engagement of the α1/α6 trigger site (2), followed by discrete structural changes, including α1-α2 loop displacement, 6A7 epitope exposure, BAX BH3 exposure, and α9 release (3). BAX is associated with the mitochondrial outer membrane that requires α9 release and exposure of the C-terminal BH3 pocket (4). Further BH3-mediated activation and BAX integration in the mitochondrial outer membrane into an undefined homo-oligomeric pore promotes release of mitochondrial apoptogenic factors such as cytochrome c (5). The autoinhibited dimeric BAX suppresses the initiation of structural changes in the cytosol by stabilizing the α1-α2 loop conformation, the 6A7 epitope and α9 in the inactive conformation (6). BH3-mediated interaction can disrupt the dimeric BAX to monomeric BAX before it induces the BAX activation pathway (7).
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions