An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway

Abstract

Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition.

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 BAX, and BAX 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-X_L, 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) Size-exclusion chromatography (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 SAHB_A 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.

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 violet for each BAX protomer. 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. Dotted line represents the missing α1–α2 loop residues that have weak electron density. (B) Ribbon representation of each BAX protomer in view related to the representation in (A) at 90 degrees 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 (grey) within the BAX dimer. Views centered on helix α5 (top view) and the N-terminal activation site (right). See also Figure S2.

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) 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.

Figure 4. Structural insights suggest a BAX auto-inhibition mechanism

(A) Structural comparison of the BAX:α9 interaction from the BAX dimer structure and BAX:BIM BH3 interaction (PDB ID: 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, L27 forming interactions with conserved hydrophobic residues F159, 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.

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). (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 SAHB_A. Reduction of the internally crosslinked BAX 4M mutant using 20 µΜ DTT followed by incubation with BIM SAHB can induce the oligomerizaton of the BAX 4M. (E) Quantification of % BAX monomers and % 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.

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 values <0.05 for BAX mutants compared to 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 staurosporine (STS) treatment at 0.5 µΜ or 1 µΜ for 12h. (C) Caspase 3/7 activation assays of DKO MEFs stably transduced with human BAX WT or mutants upon 1 µΜ STS treatment at 3, 6 and 12h. (D) BAX WT and BAX P168G cells were treated with 1 µΜ STS for 6 h; BAX E75K cells were treated for 3 h due to faster cell death. Cytosolic and mitochondrial fractions were separated and analyzed by SEC. 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.