1. Volume 30, Issue 3, Pages 369-380 (May 2008)
To Trigger Apoptosis, Bak Exposes Its BH3 Domain and Homodimerizes via BH3:Groove Interactions 
Grant Dewson, Tobias Kratina, Huiyan W. Sim, Hamsa Puthalakath, Jerry M. Adams, Peter M. Colman, Ruth M. Kluck 
Molecular Cell 
Volume 30, Issue 3, Pages (May 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Random Mutagenesis Reveals Residues Required for Bak Proapoptotic Function
(A) Bak LOF mutations identified by a yeast screen of random mutants lie in the region encompassing α helices 2–5. The three Bcl-2 homology (BH) domains and transmembrane domain (TM) are indicated, as are the α helices defined by the crystal structure (2IMS) (Moldoveanu et al., 2006). To align Bak with other Bcl-2 proteins, we have used α1′ and α2 to designate regions previously defined, respectively, as α2 and the first three turns in α3 (Moldoveanu et al., 2006).
(B) Location of LOF mutations in relation to nonactivated Bak. The surface of nonactivated Bak (2IMS) (Moldoveanu et al., 2006) is represented with each LOF mutation highlighted: BH3 domain (orange), outside the BH3 domain (yellow), or destabilizing (pink). To indicate the hydrophobic groove, the Bak BH3 peptide (red) is also indicated (based on alignment of Bak with the Bcl-xL:BakBH3 structure [1BXL] [Sattler et al., 1997]).
(C) Bak mutants fail to mediate apoptosis in mammalian cells. The Bak mutants were stably expressed in DKO MEFs, and expression was assessed by immunoblotting for Bak (aa 23–38). Cell death induced by etoposide or UV is the mean ± SEM of three independent experiments.
(D) Bak mutants still kill in the presence of WT Bak or WT Bax. HA-tagged Bak variants were transiently expressed in 293T cells, and expression was assessed at 24 hr by immunoblotting with an anti-HA antibody. Cell death at 48 hr is mean ± SEM of two independent experiments. Although the HA-tagged proteins appeared as doublets when blotted with antibodies to either HA (bottom panel) or Bak (data not shown), the untagged proteins induced equivalent death (Figure S3).
(E) The mutants are membrane integrated. Membrane fractions from untreated DKO MEFs expressing either WT Bak or Bak variants underwent carbonate extraction, and carbonate-sensitive (S) and resistant (P) fractions were immunoblotted for Bak. Reprobing for cytochrome c confirmed membrane disruption by carbonate treatment.
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3. Figure 2
Conformation of Bak Mutants before and after Apoptotic Signaling
(A and B) Most mutants expose an N-terminal epitope only after an apoptotic stimulus. (A) DKO MEFs expressing Bak variants were untreated or treated with UV and then immunostained with the conformation-specific antibody Ab-1 (solid line), or secondary antibody alone (broken line).
(B) Flow cytometry data in (A) and three replica experiments were quantified as the percentage of cells with Ab-1 epitope exposure (right of the marker in [A]) following UV. Data are mean ± SD of the four independent experiments.
(C) Trypsin proteolysis of Bak. Membrane fractions were incubated with or without tBid prior to limited trypsin proteolysis, and cleavage products were detected by immunoblotting for the Bak BH3 domain (G-23). Data are representative of three independent experiments.
(D) Most mutants lose intramolecular C14:C166 crosslinking after an apoptotic stimulus. DKO MEFs expressing Bak variants were untreated or treated with UV, and membrane fractions were exposed to the cysteine crosslinker BMH and immunoblotted for Bak (aa 23–38). The faster migrating intramolecular crosslinked monomer indicates nonactivated Bak (MX).
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4. Figure 3 Bak LOF Correlates with Impaired Homo-oligomerization
(A) Essentially all WT Bak changes conformation and homo-oligomerizes following tBid treatment. Membrane fractions from DKO MEFs expressing WT Bak were untreated or treated with tBid prior to induction of disulphide bonding (CuPhe) or treatment with cysteine crosslinkers (BMH or BMOE) to induce intramolecularly linked monomers (MX) and intermolecularly linked dimers (D). Bak in which the cysteines are not disulphide bonded or crosslinked runs as a monomer (M). Samples were separated by SDS-PAGE under nonreducing or reducing conditions. (For BMH and BMOE samples run under nonreducing conditions, see Figure S5B.) The minor band of dimerized Bak observed in lane 2 (but not in other experiments) likely reflects a small proportion of cells that underwent apoptosis prior to isolation of the membrane fractions.
(B) Mutants have impaired homo-oligomerization. Membrane fractions were untreated or treated with tBid prior to disulphide bonding with CuPhe. Bak activated by tBid that fails to form oligomers appears as a monomer (M).
Samples in (A) and (B) were immunoblotted for Bak (aa 23–38).
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5. Figure 4 Bak Proapoptotic Function Requires Exposure of Its BH3 Domain
(A and B) A Bak BH3 epitope is exposed during, but not after, tBid treatment. Membranes from DKO MEFs expressing WT Bak (A) or from mouse liver mitochondria (MLM) (B) were incubated with or without tBid. Antibodies (4B5 or Ab-1) were added after or during the tBid incubation, Bak immunoprecipitated in 1% digitonin or Triton X-100 (TX), and samples were immunoblotted for Bak (NT). # indicates IgG light chain.
(C) BH3 exposure following apoptotic signaling in cells. DKO MEFs expressing either WT Bak or Bak G126S were untreated or treated with actinomycin D and lysed in either digitonin (dig) or Triton X-100 (TX). Cell lysates were immunoprecipitated with 4B5 or AG2 antibody (upper panels), or treated with CuPhe (lower panel). Samples were immunoblotted for Bak (aa 23–38).
(D) An antibody against the Bak BH3 domain inhibits dimerization in MEF mitochondria. Membranes from DKO MEFs expressing WT Bak were incubated with tBid, and with or without antibodies (4B5, Ab-1, AG2), prior to treatment with CuPhe and immunoblotting for Bak (aa 23–38).
(E) An antibody against the Bak BH3 domain inhibits Bak dimerization and cytochrome c release in MLM. MLM were incubated with or without tBid, and with increasing concentrations of 4B5 or AG2 (μg per 50 μl incubation). Samples were treated with the BMH crosslinker and immunoblotted for Bak (NT), or pellet (P) and supernatant (S) fractions were collected and immunoblotted for cytochrome c.
(F) Proximity of Bak α1 and α2 residues. Schematic of Bak α1 (blue) and α2 (red) in nonactivated Bak, showing the intramolecular β carbon distances (Å) for three pairs of residues.
(G) Bak Y41C, A79C, and Y41C/A79C mutants retain WT Bak function. DKO MEFs stably expressing the indicated Bak variants were treated with UV. Data are mean ± SEM of three independent experiments.
(H) Crosslinking of Y41C and A79C in nonactivated Bak tethers α1 to α2. Membranes from DKO MEFs expressing WT Bak or Y41C/A79C were treated with or without CuPhe and immunoblotted for Bak (aa 23–38).
(I) Constraining α2 inhibits Bak proapoptotic function. Membranes from DKO MEFs expressing the indicated cysteine mutants were treated where indicated with CuPhe prior to treatment with tBid. Pellet (P) and supernatant (S) fractions were immunoblotted for cytochrome c.
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6. Figure 5
BH3 Mutation Partially Rescues LOF Mutation in the Hydrophobic Groove
(A) LOF Bak mutations are in well-conserved residues. The mutations involve residues (bold) that show conservation (gray) with multidomain relatives in the BH1 (hydrophobic groove) domain and with BH3-only proteins in the BH3 domain. Also indicated are the conserved glycine (G, yellow) and asparagine (N, red) (see [B]).
(B) Bak G126 and N86 are in close proximity in a BH3:groove interaction. The structures of Bak (Moldoveanu et al., 2006) and Bcl-xL:Bim (Liu et al., 2003) were structurally aligned to show Bak (surface) and the Bim BH3 (red tube) and the predicted proximity of Bak G126 (yellow) with the Bak N86 equivalent (BimEL N158, red).
(C) Mutation in BH3 (N86G) rescues proapoptotic function of groove mutant G126S. Lysates of DKO MEFs expressing the indicated Bak variants were immunoblotted for Bak (aa 23–38) or β-actin as a loading control (lower panels). Also, cells were treated with actinomycin D (0.1–1 μM) for 24 hr, or with UV (100 J/m2) and incubated for up to 24 hr. Cell death is expressed as mean ± SEM of three or more independent experiments.
(D) N86G partially rescues G126S oligomerization. Membrane fractions from cells used in (C) were untreated or treated with tBid prior to treatment with BMH and immunoblotting for Bak (aa 23–38).
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7. Figure 6
Cysteines Placed in Bak BH3 and Groove Can Disulphide Bond after Apoptotic Signaling
(A) Predicted proximity of BH3 domain and groove residues. The Bak BH3 (aa 69–88; red) and hydrophobic groove α3 and α4 (aa 86–120; green) are based on an alignment of the Bak and Bcl-xL:Bim structures as in Figure 5B. Calculated molecular distances (Å) between β carbons of particular residues are indicated. Others not shown are as follows: 11.4 Å for G72:Y110; 14.0 Å for G72:H99.
(B) Cysteine mutants retain proapoptotic function. The indicated Bak variants were tested as in Figure 4G. Cell death is the mean ± SEM of three or more independent experiments.
(C) BH3 residues are juxtaposed with α3 and α4 residues after tBid treatment. Pairs of FLAG- and HA-tagged single-cysteine Bak variants were coexpressed in DKO MEFs. Membrane fractions were treated with tBid and then with CuPhe. Samples were immunoprecipitated for the FLAG epitope and assessed for recovery (middle panel) and coprecipitated HA-tagged variants (upper panel). Disulphide-bonded dimers (D) and non-disulphide-bonded but immunoprecipitated variants (M) are indicated. Cell lysates taken prior to CuPhe addition indicate HA-Bak and FLAG-Bak protein levels (lower panels).
(D) BH3:groove interaction after apoptotic signaling in cells. Cells used for (C) were left untreated or treated with actinomycin D, then membrane fractions were treated with CuPhe and analyzed as in (C). Asterisk (∗), lower band may be due to degradation of the HA-H99C mutant.
(E) BH3:groove dimers form only after tBid treatment. Membranes from DKO MEFs coexpressing the indicated pairs of FLAG- and HA-tagged single-cysteine variants were treated with or without tBid prior to CuPhe treatment and immunoprecipitated as in (C). Asterisk (∗), nonspecific band.
(F) Triton X-100 alters Bak conformation but does not induce dimerization. Membranes from DKO MEFs expressing WT Bak were untreated, or treated with tBid or 1% Triton X-100, prior to treatment with CuPhe and immunoblotting for Bak (aa 23–38).
Data in (C) to (F) are representative of two or more independent experiments.
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8. Figure 7 Models of Bak Oligomerization during Apoptosis
(A) Schematic of BH3 exposure and binding to the hydrophobic groove during Bak activation and oligomerization. Bak α helices 1–4 are depicted as in nonactivated Bak (Moldoveanu et al., 2006), with α1 on the opposite face to α3 and α4 (top). Upon activation, α2 (the BH3 domain) is everted, perhaps in association with extension of the α1-α2 loop (middle left). (That α1 also changes conformation is suggested by exposure of the Ab-1 epitope in Figures 2A and 4A.) The exposed BH3 domain then can bind to the hydrophobic groove of another Bak molecule to form a Bak dimer, perhaps via a symmetric interaction (bottom) (see [B]), or to the hydrophobic groove of the prosurvival proteins Bcl-xL and Mcl-1, if either is unoccupied (right).
(B) Three models by which Bak could form dimers and higher order oligomers. Our data favor the symmetric dimer model (see the Discussion).
Molecular Cell  , DOI: ( /j.molcel )
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