1. Volume 152, Issue 3, Pages 519-531 (January 2013)
Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis 
Peter E. Czabotar, Dana Westphal, Grant Dewson, Stephen Ma, Colin Hockings, W. Douglas Fairlie, Erinna F. Lee, Shenggen Yao, Adeline Y. Robin, Brian J. Smith, David C.S. Huang, Ruth M. Kluck, Jerry M. Adams, Peter M. Colman 
Cell 
Volume 152, Issue 3, Pages (January 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Dimerization of BaxΔC21 Promoted by Octylmaltoside or BH3 Peptides
(A) Gel filtration profile of BaxΔC21 treated with CHAPS, BimBH3, octylmaltoside (OM), or CHAPS and BimBH3.
(B) Blue native PAGE of BaxΔC21 showing dimerization with OM or with CHAPS plus BimBH3 peptide. The Bim-induced dimer probably is more diffuse than the OM one because the peptide partially disassociates during the run. The faster migrating monomer produced by BimBH3 alone may be indicative of an altered monomer conformation.
(C) Blue native PAGE of BaxΔC21, showing dimers in the presence of CHAPS and certain BH3 peptides, but not Noxa or Bad.
(D) SDS PAGE showing exposure of the N-terminal epitope 49F9 by immunoprecipitation of BaxΔC21 during treatment with octylmaltoside. As noted later, the epitope is exposed only transiently during the transition from monomer to dimer (Figure 5). Asterisk indicates immunoglobulin light chain. Data are representative of three experiments.
See also Figure S1.
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4. Figure 2 Structure of BidBH3:BaxΔC21 Complex
(A) Dimer of BaxΔC21, colored rainbow (blue N terminus to red C terminus) in complex with BidBH3 (magenta), illustrating domain swapping of helices 6–8 (colored orange and red) across the dotted line.
(B) BidBH3 peptide (magenta) projects four canonical hydrophobic residues (h1 through h4) into the canonical binding groove of Bax (green). The h0 residues I82 and I83 make additional interactions with the Bax groove. Bid D95 forms a salt link to Bax R109 (cyan). Other Bax side-chains shown (cyan) are R94 (close to Bid I83) and I66 and Y164 (both close to Bid M97).
See also Figure S2.
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5. Figure 3 Characteristics of Activator BH3 Domains
(A) BH3 peptide sequences.
(B) Blue native PAGE assays of CHAPS/peptide-induced dimerization of BaxΔC21.
(C) Release of fluorescent dextran from liposomes exposed to full-length Bax and BH3 peptides. Note especially loss of function in Bid I82A/I83A and gain of function in Noxa C25I and Bad mut4/mini. Error bars represent SEM of at least three independent experiments.
(D) Noxa mutant C25I and Bad mut4/mini, but not wild-type peptides nor NoxaC25I with Ala at h0, release cytochrome c from mouse liver mitochondria derived from Bak−/− mice and reconstituted with recombinant wild-type, full-length Bax. SN, supernatant. See also Table S2. (Bad mut4 was not included in liposomes or MLM experiments as it permeabilized these in the absence of Bax.)
(E) The Bax groove mutant R109D is not dimerized by CHAPS with wild-type BaxBH3 peptide but, rather, is with the Bax peptide D68R, which cannot dimerize wild-type Bax. Data are representative of three experiments.
See also Figure S3.
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6. Figure 4 Comparison with Structures of Bcl-xL:BH3 Complexes
(A) Cavity (orange, 140 Å3) in the hydrophobic core of BidBH3:Bax complex between α2, α5, and α8. The cavity is adjacent to Bax α2 residues L63 and I66 (side chains shown in gray). The largest comparable cavity in BimBH3:Bcl-xL is 20 Å3.
(B) Overlay of BidBH3:Bax with BimBH3:Bcl-xL and zoom showing relative displacement of the Bax α2 and the contacts between hydrophobic residues on the BH3 domains of protein (h3 residues Bcl-xL A93/Bax I66) and peptide (h4 residues Bid M97/Bim F159).
See also Figure S4.
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7. Figure 5 Bax Core and Latch Domains Separate during Apoptosis
(A) Core/latch domain-swapped dimer of BaxΔC21 (chain A rainbow as in Figure 2A; chain B, gray). The disordered loop between α1 and α2 introduces an uncertainty in assigning α1 to chain A or B.
(B) A putative intermediate in transition from monomeric BaxΔC21 to the structure in (A), exposing the 49F9 epitope (magenta). V121 and I136, where cysteine substitutions were made to tether α5 to α6, are labeled. Some unfolding of the latch (and to a lesser extent the core) is expected during the transition.
(C) The α5–α6 tether forms on mitochondria only before activation with tBid. Membrane fractions were treated with or without tBid prior to induction of disulfide-linkage with copper(II)(1,10-phenanthroline) (CuPhe).
(D) The α5–α6 tether prevents Bax function. Membrane fractions were treated with CuPhe prior to tBid-induced cytochrome c release. SN, supernatant fraction.
(E) The α4–α5 tether persists after activation with tBid. Membrane fractions were treated as in (C).
(F) The α4–α5 tether does not affect function. Membrane fractions were treated as in (D). Data in (C)–(F) are representative of three experiments with mitochondria from Bax−/− Bak−/− cells stably expressing the indicated Bax variants.
(G and H) (G) BaxΔC21 with the mutation V121C/I136C still binds BidBH3 peptide, both before and after oxidation (IC ± 32 nM and 159 ± 45 nM, respectively; SD, n = 3), and in (H), the reduced form can be dimerized by CHAPS plus BimBH3, but the oxidized form cannot.
See also Figure S5.
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8. Figure 6 The BH3-in-Groove Symmetric Dimer Formed by Bax(α2–α5)
(A) Each polypeptide is colored rainbow as in Figure 5B, cyan at the N terminus of α2 through yellow at the C terminus of α5. The helices of one polypeptide are labeled. E69 and R65 are indicated in the left view, and aromatic residues concentrated on one surface are indicated in the right view, the putative “membrane view.”
(B) Overlay of one half of the above dimer (α2 from one polypeptide, α3–5 from the other) with the BaxBH3:BaxΔC21 complex (see Figure S2B).
(C) Bax E69C can be crosslinked on mitochondria. Membrane fractions from Bax−/− Bak−/− cells stably expressing the indicated Bax S184L variants were treated with or without tBid prior to crosslinking with BMOE. Note that R65C one turn away on helix α2 cannot crosslink.
See also Figure S6.
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9. Figure 7
Models Demonstrating Our Conclusions Regarding BaxΔC21 Activation and Bax(α2–α5) Dimerization and Their Implications for Bax Anchored to the MOM
(A) (i) BaxΔC21 is monomeric in the absence of detergents or CHAPS plus BH3 peptides. (ii) Binding of an activator BH3 peptide into the Bax canonical groove unlatches the core domain (α1–α5). (iii) In the absence of an orienting membrane tether, two such unlatched molecules combine (“head to tail”) to form the core/latch dimer. Note the change in orientation of the core in these figures once the latch is released (i to ii). The core alone instead forms the on-pathway BH3-in-groove symmetric dimer.
(B) With MOM-anchored native Bax, we propose that activator BH3 domains bind into the groove and unlatch the structure as in (A), releasing the Bax BH3 domain and allowing it to compete for the groove of neighboring Bax molecules on the MOM. The final complex, the BH3-in-groove symmetric Bax dimer, is favored due to the stability afforded by dimerization. In that dimer, the positions of the latch domain and the membrane-anchoring α9 are unknown. The dimer’s lipophilic α4–α5 surface may engage the MOM and promote its permeabilization.
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10. Figure S1
Characterization of Conformation-Specific Anti-Bax Antibody 49F9 Used for Immunoprecipitation in Figure 1D, Related to Figure 1
(A) Epitope mapping of 49F9, which was raised against a peptide corresponding to residues 12 to 24 of human Bax. Histograms show immunoreactivity of 49F9 (1 μg/ml) against biotinylated hBax peptides.
(B) 49F9 epitope exposure in response to etoposide. Bax−/−Bak−/− MEF, reconstituted with hBax, were treated or not with etoposide and the heavy membrane fractions solubilized in TX-100 or CHAPS. Bax was then immunoprecipitated with 49F9 and detected by Western blot with anti-Bax N-20. The epitope for 49F9 overlaps that for 6A7 (residues 13-19) (Hsu and Youle, 1997).
(C) 49F9 epitope exposure revealed by SDS PAGE following immunoprecipitation of BaxΔC21 during treatment with BimBH3 and CHAPS. The panels are from the same gel. The much lower exposure with BimBH3 than OM may be because their transitory ‘unlatched forms’ (see Figure 5B) differ slightly in structure (e.g., the disposition of α1), or because the ‘unlatched form’ persists longer with OM before turning into the domain-swapped dimer, in which the epitope is reburied.
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11. Figure S2
Structure of BaxΔC21 in Complex with BH3 Peptides, Related to Figure 2
(A) 2FO-FC (1σ blue) and FO-FC (3σ green) stereo electron density maps of a portion of the BidBH3:BaxΔC21 complex. The maps are phased on the molecular replacement solution for Bax alone. Overlaid on these unbiased maps is the final refined structure of portion of the BidBH3.
(B) The BaxBH3 peptide (magenta) projects hydrophobic residues into the canonical groove of BaxΔC21 (green) in the same way as shown for the BidBH3 peptide in Figure 2B, and in the same way as BH3 peptides engage pro-survival proteins. For example, BaxBH3 h2 residue (L63) and Bid BH3 h2 residue (L90) both engage Bax residues M99 and F116, just as Bim h2 residue L152 engages Bcl-xL residues L130 and F146 (Lee et al., 2009; Liu et al., 2003). Note proximity of R109 of Bax to D68 of the BaxBH3 peptide, used in charge-swap experiments (Figure 3E).
(C) Identical view to (B) but with BaxΔC21 represented as a surface illustrating the five pockets that accommodate the hydrophobic residues h0 through h4 of the BaxBH3 peptide.
(D) Proximity of R94 of Bax and T56 of bound BaxBH3, consistent with cross-linking studies on Bax homo-oligomers in dying cells (Dewson et al., 2012).
(E) C62 of Bax (mutated to S62 in this work) is 21.5 Å from C62 of bound Bax BH3, consistent with a reported distance of 24 Å between C62 residues as determined by electron paramagnetic resonance (EPR) measurements on activated Bax in liposomes (Bleicken et al., 2010) (See also Figure S6E).
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12. Figure S3 Features of Activator BH3 Domains, Related to Figure 3
The mutants described here and in Figure 3 were designed to assess the role in Bax activation of (i) the four canonical hydrophobic residues h1 to h4, (ii) the N-terminal ‘h0’ residues, (iii) the Arg-Asp salt bridge and (iv) BH3 residues C-terminal to h4.
(A) Biacore measurements (with 95% confidence interval from a representative experiment done in triplicate) of peptide binding to detergent-activated BaxΔC21.
(B) Inhibition curves for peptides displaying high or moderate inhibition (titration performed from 2.93 nM to 3 μM).
(C) Inhibition curves for Noxa wt BH3 and Noxa C25I BH3 peptides. As they bound more weakly than those in (B), they were titrated from 47 nM to 24 μM.
(D) Inhibition curves for peptides showing negligible inhibition of the binding of BaxΔC21 to BimBH3 peptide (titration from 2.93 nM to 3 μM). Error bars in (B), (C) and (D) represent SEM of triplicate data used to calculate the representative IC50 values in (A).
(E) Location of BimBH3 ‘h0’ and h1 residues in the proposed alternative Bax binding site, the α1/α6 interface (Gavathiotis et al., 2008). The model depicts a stapled BimBH3 (slate) interacting with Bax (cyan) at the α1/α6 interface (PDB 2K7W). The equivalent ‘h0’ residues to I82 and I83 in Bid are P144 and E145 in Bim. P144 is not in the peptide used in the BimSAHB studies, and E145 (displayed with side chain magenta) is solvated in this model. I148 at the h1 position of Bim is shown green.
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13. Figure S4
Comparison of Prosurvival and Proapoptotic Proteins, Related to Figure 4
(A) The structure of the BaxBH3:Bax complex reveals a cavity (orange, 170 Å3) similar to that in the BidBH3:Bax complex (Figure 4A).
(B) Structural alignment of one globule of the BaxBH3:Bax dimer with the BaxBH3:Bcl-xL complex (PDB:3PL7) (Czabotar et al., 2011). The displacement of α2 of Bax is similar to that in the BidBH3:Bax structure (Figure 4B).
(C) Sequence alignment of the α2 to α5 region of Bax with the equivalent regions of pro-survival proteins. Grey highlights mark the four signature hydrophobic residues h1 through h4, which together with a conserved aspartyl residue between h3 and h4 (bold) characterize a BH3 domain. An asterisk marks the h3 position, which in Bax and Bak is Ile and in pro-survival proteins is Ala or Val (see Figure 4B). Underlined sequences are the crystallographically determined helical segments. BH3 domains for other pro-apoptotic proteins are also shown.
(D) Mutation of the BimBH3 and BidBH3 peptide at the h4 position (F159A and M97A, respectively) reduces their ability to promote dimerization of Bax wild-type and particularly of Bax I66A. Gel filtration plots of samples incubated for 1 hr are displayed.
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14. Figure S5
Characterization of Unliganded BaxΔC21 and the Unliganded BaxΔC21 Domain-Swap Dimer, Related to Figure 5
(A–C) NMR data from monomeric BaxΔC21, showing that the largest differences between full-length Bax and BaxΔC21 occur in the vicinity of the Bax groove occupied by helix α9 as shown in panel D.
(A) 2D 1H-15N HSQC spectrum of BaxΔC21 (C62S, C126S) at 305K. C-terminal residues GSS, which originate from the vector, are Resonances from S16 and A54, invisible at the current contour level, are indicated with x in red.
(B) Expanded central region of (A).
(C) Weighted average differences between backbone 1HN and 15N chemical shifts of BaxΔC21 and those of full length Bax (Suzuki et al., 2000), Δδav, plotted against residue number of BaxΔC21 (Δδav = {[(Δδ (1HN))2+(0.2 × Δδ (15N))2]/2}1/2).
(D) Overlay of one globule of BaxΔC21 extracted from the core/latch dimer (orange) with the NMR structure of full-length Bax (slate, PDB code 1F16) (Suzuki et al., 2000). The globule comprises the core domain from chain A and the latch domain from chain B as shown in Figure 5A. In Bax ΔC21, the C-terminal end of α2 has collapsed against α5, and α3 against α4, occupying space filled by α9 in full-length Bax. Thus, the groove is nascent in the absence of α9.
(E) Overlay of one globule of apo BaxΔC21 extracted from the core/latch dimer (orange) with one globule from the BidBH3:Bax complex (Figure 2B) (magenta and green). Groove remodeling involves shifts in α2 and α3 to accommodate the BH3 peptide, similar to that observed in the comparison to full-length Bax (panel D). Only the peptide-bound crystal structure contains a cavity (Figures 4 and S4).
(F) Bax cysteine mutants used in tethering experiments to test detachment of latch from core are functional. Bax−/−Bak−/− MEF reconstituted with Bax (S184L, C62S, C126S) bearing mutations to test the effect of tethering α5 to α6 (V121C to I136C) or α4 to α5 (V91C to L120C) (see Figures 5C–5H) were assessed in cell death assays by analyzing the percentage of PI-positive cells after etoposide exposure (10 μM) for 24 hr. Error bars represent SD (n = 3).
(G) Expression levels of Bax variants in panel F, reblotted for Hsp70 as loading control.
(H) Tryptophan region of the 2D 1H-15N HSQC spectrum of BaxΔC21 (C62S, C126S, V121C, I136C) at 305K treated with either DTT (red) or CuPhe (purple) indicating that the mutant is folded even when it is oxidized and α5 is tethered to α6. Overlaid in blue is the same region from (A) with assignments indicated.
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15. Figure S6 BH3-in-Groove Symmetric Dimers, Related to Figure 6
(A and B) Maps obtained after molecular replacement using only GFP as a search model against diffraction data from GFP-Bax(α2-α5) crystals (2Fo-Fc, blue, contoured at 0.8 σ; Fo-Fc, green, contoured at 2.5 σ). (A) Maps with only the GFP component from the final model displayed (cartoon format; colored according to chain, cyan and magenta). (B) Maps from panel A with both the GFP and Bax(α2-α5) from the final model displayed.
(C) Tetramer of GFP-Bax(α2-α5) within the crystal lattice. In that lattice GFP dimer interfaces do not influence formation of Bax BH3-in-groove dimer interfaces.
(D) GFP-Bax(α2-α5) dimer colored according to B-factor (Blue to Red, cold to hot), demonstrating higher atomic displacement parameters of Bax(α2-α5) than GFP. Average B-factor for GFP components was 103 Å2 but 176 Å2 for Bax components.
(E) Agreement of the Bax BH3-in-groove dimer structure with other experimental evidence. The observed proximity of R94 (blue) to T56 (yellow) is consistent with cysteine linkage by CuPhe in oligomeric Bax (Dewson et al., 2008, 2012). The distance between symmetrical E69 residues (9.3 Å) is similarly consistent with crosslinking by BMOE (Figure 6C). The spacing between C62 residues (replaced with Ser in this construct) agrees with that observed in EPR measurements of activated Bax in liposomes (24 Å) (Bleicken et al., 2010). M74 does not contribute to the dimer interface, consistent with evidence that M74 mutants remain able to homo-oligomerize (Czabotar et al., 2011).
(F) Gel filtration plots for GFP-Bax(α2-α5) and GFP-Bax(α1-α5), showing that a tetramer predominates in both. (The GFP for the GFP-Bax(α2-α5) construct bears the unintentional mutation A206N, whereas that in GFP-Bax(α1-α5) is wild-type.)
(G) Bax cysteine mutants used in cross-linking experiments are functional. Bax−/−Bak−/− MEF were reconstituted with Bax (S184L, C62S, C126S) bearing mutations to assess for cross-linking between adjacent Bax BH3 domains (Figure 6C). Bax function was assessed in cell death assays by analyzing the percentage of PI-positive cells after etoposide exposure (10 μM) for 24 hr. Error bars represent SD (n = 3).
(H) Expression levels of mutants in panel G, reblotted for Hsp70 as loading control.
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