Interactions between BCL-2 and BAX BH3 peptides. (Left) The structure of BCL-2 bound to the 31-mer BAX peptide. BCL-2 and the BAX peptide are in pink and green, respectively. (Right) Binding affinities. Each of the three indicated BAX peptides was titrated into the BCL-2 solution and the deduced K_D values are shown in the table. The sequences of the BAX peptides are listed below, with the conventionally defined BH3 domain marked in red.

BAX(AAA) has impaired affinity for BCL-2 and is functionally active. (A) Cell-based protein-binding assay. The indicated proteins were transiently expressed in 293T cells. The amount of BCL-2 or endogenous BAX associated with these proteins was detected by immunoprecipitation using antibody against Flag followed by immunoblot analysis using antibody against BCL-2 or BAX. BAX(AAA) fails to bind endogenous BCL-2, but associates with endogenous BAX. Coprecipitation of Flag-BAX and endogenous BAX is conceivably through homooligomerization. Transfection with empty vector as a control is indicated by “Vector” throughout the figures. (B, C) Apoptosis assays. Bax^−/− Bak^−/− MEFs stably expressing the indicated proteins were treated with etoposide (100 μM) for 36 h, and cell viability or the ratio of apoptotic cells was assessed by PI staining (B) or Annexin V staining (C) and flow cytometry. Values are the average of three independent assays ± standard deviations. The two assays clearly show that BAX(AAA) induces apoptosis as efficiently as wild-type BAX. Further corroboration of these results is shown in Supplementary information, Figure S3. Expression levels of the BAX proteins are shown in Supplementary information, Figure S4A.

Interactions between antiapoptotic BCL-2 proteins and BH3-only proteins. (A) Binding affinities. ITC analysis was performed by titrating the 36-mer BH3 peptides derived from the indicated BH3-only proteins (0.2 mM) into the five antiapoptotic BCL-2 proteins (10 μM). (B) A plot of the measured K_D values on a log scale. The diagram includes the K_D values measured for the BAX and BAK peptides (Figure 1A). The dash marks in front of the names of the proapoptotic proteins (in black or red letters) are positioned at the measured binding affinities of their BH3 peptides for the indicated antiapoptotic BCL-2 proteins (in blue letters).

Interactions between antiapoptotic BCL-2 proteins and BAX/BAK. (A) Binding affinities. ITC analysis was carried out by titrating the 36-mer BH3 peptides of BAX and BAK (0.2 mM) into the indicated antiapoptotic BCL-2 proteins (10 μM). The K_D values were deduced from curve fittings of the integrated heat per mol of added ligand and are listed in the tables. Two representative ITC runs are shown below. (B) Endogenous interactions. Whole cell lysates of 293T cells were used for immunoprecipitation with control rabbit serum, anti-BAX or anti-BAK antibody followed by immunoblotting with antibodies against the indicated BCL-2 proteins. (C) The BAX peptide preferentially binds to BCL-2 over BCL-X_L. The BAX peptide (5 μM) was incubated together with BCL-2 (lane 2), BCL-X_L (lane 4) or both the proteins (lane 6) at the same concentration (5 μM), and the mixture was visualized on a native gel. Free BCL-2 is unobservable, while most of BCL-X_L remains free. (D) A1 binds both endogenous BAX and BAK. HA-tagged A1 was transiently expressed in 293T cells and whole-cell lysates were used for immunoprecipitation with control rabbit serum, anti-BAX or anti-BAK followed by immunoblotting with anti-HA, anti-BAX or anti-BAK antibody.

Structural and mutational analysis of the interaction between BCL-2 and the BH3 domain of BAX. (A) Intermolecular interactions. Two surface models of BCL-2 with the bound BAX peptide in green, showing the hydrophobic (left) and the ionic interactions (right) separately. The residues engaged in those interactions are shown in sticks and labeled. The surface coloring scheme is as follows: yellow for Val, Leu, Ile, Tyr, Phe, Trp, Met and Ala; blue for Lys, Arg and His; red for Glu and Asp; gray for other amino acids. (B) Sequence alignment of the BH3 domains in the proapoptotic BCL-2 proteins. Conserved residues are highlighted in red (> 80% similarity) or pink (> 60% similarity) columns. The black and red arrows indicate the BAX residues shown in (A). The five consensus BH3 residues that are known to be critical for the interactions with the antiapoptotic BCL-2 family members ⁷ are indicated by asterisks. (C) Contribution of the charged residues of BAX to the binding affinity. BAX peptides (36-mer) with the alanine substitution of the indicated charged residues were titrated into BCL-2, and the deduced K_D values are listed in the table. Shown in the bottom is the ITC run for the titration of the BAX peptide with the triple substitutions.

BAX(AAA)-mediated apoptosis is barely inhibited by BCL-2 and BCL-w. (A) Bax^−/− Bak^−/− MEFs stably expressing HA-tagged BCL-2 (DKO.BCL-2) were transfected with a BAX- or BAX(AAA)-expression vector and treated with 50 μM etoposide for 36 h. The apoptotic cells were estimated by PI staining and flow cytometry. (B) At 16 h post-transfection with a plasmid expressing HA-BCL-2, the Bax^−/− Bak^−/− MEF cells stably expressing vector only, BAX, BAX(AAA) or BAX(L63A) were treated with 100 μM etoposide for 8 h, fixed, and were immunostained with anti-HA antibody (red) or 6A7 (green) followed by confocal analysis. A single representative optical section is presented. The white and yellow arrows indicate BCL-2-expressing cells and 6A7-positive cells (Act BAX), respectively. The bottom panel shows the percentage of BCL-2-transfected cells positively stained with 6A7 antibody (∼200 cells collected per sample). (C) Bax^−/−Bak^−/− MEFs stably expressing vector only, BAX, BAX(AAA) or BAX(L63A) were transfected for ectopic expression of HA-tagged BCL-w. After etoposide (100 μM) treatment for 36 h, cells were stained with Annexin V and analyzed by flow cytometry (top). Apoptotic cells are estimated and plotted (bottom). The error bars indicate standard deviations calculated from three independent assays. Expression levels of the BAX proteins and BCL-w are shown in Supplementary information, Figure S4C.

Displacement assays. For in vitro assay (A-D), the 36-mer BH3 peptides were used, which are indicated by the names of the proapoptotic BCL-2 proteins. BCL-2 was preincubated with a BH3 peptide at 1:1 molar ratio or up to 1:8 molar ratio (in C) for 1 h. A competitor peptide (in red) was added to the mixture and incubated for additional 1 h. The numbers in parentheses indicate the ratios between the peptides. The mixture was visualized on a native gel. (A) The BAD peptide hardly displaced the BIM peptide bound to BCL-2, as most of the BCL-2-BIM peptide complex remained intact up to eight-fold molar excess of the BAD peptide over the BIM peptide. (B) The BIM peptide displaced the BAX peptide completely and formed a complex with BCL-2 (lane 6). The BAD peptide displaced the BAX peptide but only slightly (lane 7). The NoxaA peptide failed to displace the BAX peptide (lane 8). (C) The BIM peptide readily displaced the BAX peptide bound to BCL-2. Formation of BCL-2-BIM peptide complex is visible even at 8:1 molar ratio between the BAX and BIM peptides. (D) The BAD peptide inefficiently displaced the BAX peptide bound to BCL-2. The BCL-2-BAX peptide complex remained even at 1:8 molar ratio between the BAX and BAD peptides. (E) Cell-based displacement assay. Flag-BIM_EL, Flag-BAD or Flag-Noxa was transiently expressed in 293T cells and the effect of the expression on the endogenous BCL-2-BAX interaction was assessed by immunoprecipitation and immunoblotting. (Left) BIM_EL sequesters BCL-2 from BAX binding; (middle) BAD binds BCL-2 and displaces BAX; (right) Noxa does not bind BCL-2 and has no effect on the BCL-2-BAX interaction. The asterisks indicate the light chain of antibody.