BAX activation is initiated at a novel interaction site

Abstract

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

Figure 1. NMR analysis of BAX upon BIM SAHB titration

a, Chemical shift changes are plotted as a function of the residue number of BAX. Residues with significant backbone amide chemical shift change (>0.01 ppm) are colored orange. The absence of a bar indicates the presence of a proline or a residue that is overlapped or below detection threshold. Error bars, mean ± s.d. b, The C^α atoms of BAX residues affected by BIM SAHB binding are shown as orange spheres. c, Surface diagram illustrating the BAX binding site. Side chains of hydrophobic, positively charged, negatively charged, and hydrophilic residues are colored yellow, blue, red, and green, respectively.

Figure 2. Orientation of BIM SAHB at the novel BAX binding site

a,Paramagnetically-labeled derivatives of BIM SAHB. b, Ratios of BAX crosspeak intensities in the presence of oxidized or reduced BIM SAHB_MTSL (I_ox/I_red) versus residue number. BAX residue intensities reduced below 0.6 are colored. c, Orientation of BIM SAHB at the BAX site. Based on an I_ox/I_red threshold of 0.6, BAX residues impacted by BIM SAHB_(A164C-MTSL), BIM SAHB_(W147C-MTSL), or both are colored in purple, green, or brown, respectively.

Figure 3. BIM SAHB directly initiates BAX activation in vitro

a, Oligomerization (a) and 6A7 immunoprecipitation (b) of BAX after BIM SAHB treatment. c, Liposomal FITC-dextran release in the presence of BAX and BIM SAHB. d, Cytochrome c release from BAX/BAK-null mitochondria upon incubation with BAX and BIM SAHB. Error bars, mean ± s.d.

Figure 4. Sequence specificity of BIM SAHB-induced BAX activation

a, BIM SAHBs with differential staple positions and their activities in BAX oligomerization (b) and BAX-mediated cytochrome c release assays (c). d, BIM SAHB_A point mutants and their activities in BAX oligomerization (e) and BAX-mediated cytochrome c release assays (f). Error bars, mean ± s.d.

Figure 5. Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosis

a, Impact of BAX K21E mutagenesis in oligomerization and b, cytochrome c release assays. c, Apoptotic response of Bax^-/-Bak^-/- MEFs reconstituted with BAX or BAX_K21E to treatment with BIM SAHB_A or BIM SAHB_A(R153D). d, Impact of BAX K21E mutagenesis on staurosporine (STS)-induced apoptosis of BAX-reconstituted DKO MEFs. Error bars, mean ± s.d.