BH3-triggered structural reorganization drives the activation of proapoptotic BAX

Abstract

BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.

Figure 1. BAX activation is initiated by ligand-induced displacement of the α1-α2 loop from a closed to an open conformation

(A) NMR analysis of ¹⁵N-BAX upon BIM SAHB titration revealed conversion of BAX from a “closed-loop” to an “open-loop” conformation, implicating α1-α2 loop displacement as the initial conformational change upon BH3-triggered BAX activation. (B) Noncovalent interactions between select α1-α2 loop and α6 residues reinforce the “closed-loop” conformation of inactive BAX (Suzuki et al., 2000). (C) The L45 (α1-α2 loop) and M137 (α6) noncovalent interaction pair was replaced with cysteines to generate a disulfide linkage that covalently locks the α1-α2 loop in the closed conformation. (D) BIM SAHB-induced BAX oligomerization was monitored by size-exclusion chromatography. Oxidized wild-type BAX monomer (0.5 mM GSSG) exhibited time-dependent oligomerization in response to equimolar BIM SAHB treatment, whereas oxidized BAX(L45C-M137C) monomer showed no response. The addition of 100 mM BME to BAX(L45C-M137C) restored BIM SAHB-induced oligomerization, but had no effect in the absence of triggering ligand. (E) Cytochrome c release assays were performed using mitochondria isolated from Alb-cre^posBax^flox/-Bak^−/− mice. Oxidized (0.5 mM GSSG) BAX(L45C-M137C) (100 nM) did not induce mitochondrial cytochrome c release in response to BIM SAHB treatment. However, the addition of BME (100 mM) to BAX(L45C-M137C) restored BIM SAHB-triggered cytochrome c release to wild-type levels. The addition of 0.5 mM GSSG or 100 mM BME to wild-type BAX had no independent effect on the induction of cytochrome c release by BIM SAHB. See also Supplementary Figure 1.

Figure 2. Release of α9 from its hydrophobic binding pocket is required for BH3-triggered BAX activation

(A) The Cα atoms of ¹⁵N-BAX residues affected by BIM SAHB titration up to a ratio of 1:6 BAX:BIM SAHB are represented as orange spheres in the ribbon diagram. A side view of the BAX structure, rotated 90° from the α1/α6 trigger site (purple), demonstrates the series of α9 residues (red) with significant backbone amide chemical shift changes (calculated significance threshold ≥0.02 p.p.m.) The corresponding ¹H-¹⁵N HSQC spectra demonstrate the dose-responsive chemical shift changes of BAX α9 residues (e.g. T174, L187, and K189) in response to BIM SAHB titration at 1:2, 1:4, and 1:6 ratios of BAX:BIM SAHB compared to unliganded BAX. (B) BAX residues A112 of helix α5 and V177 of helix α9 were mutated to cysteines to generate the α9-tethered BAX(A112C-V177C) construct, in which α9 is covalently locked into its binding pocket (green). (C) Oxidized BAX(A112-V117C) monomer (0.5 mM GSSG) was not activated by equimolar BIM SAHB (1:1), whereas wild-type BAX monomer exposed to oxidant underwent BIM SAHB-triggered oligomerization. The addition of BME to BAX(A112C-V117C) monomer completely restored BIM SAHB-induced oligomerization, an effect that was ligand-dependent, as the addition of BME alone did not induce activation. (D) Upon exposure to isolated mitochondria, BAX(A112C,V117C) (100 nM) remained in the soluble fraction irrespective of redox status. BIM SAHB triggered mitochondrial translocation of reduced BAX(A112C,V117C), but not oxidized BAX(A112C-V117C). (E) Correspondingly, BAX(A112C,V117C) (100 nM) induced mitochondrial cytochrome c release only when reduced with BME and treated with BIM SAHB. See also Supplementary Figure 2.

Figure 3. The BAX BH3 helix is exposed upon ligand-triggered direct BAX activation

(A, B) Measured chemical shift changes of ¹⁵N-BAX(A112C-V117C) (A) and ¹⁵N-BAX(P168G) (B) upon BIM SAHB titration up to a ratio of 1:2 BAX:BIM SAHB are plotted as a function of BAX residue number. The weighted average chemical shift difference Δ at the indicated molar ratio was calculated as $\sqrt{\left\{{\left(\Delta \text{H}\right)}^2+{\left(\Delta \text{N}/5\right)}^2\right\}/2}$ in p.p.m, as previously reported (Gavathiotis et al., 2008). The absence of a bar indicates no chemical shift difference, or the presence of a proline or residue that is overlapped or not assigned. The significance threshold for backbone amide chemical shift changes was calculated based on the average chemical shift across all residues plus the standard deviation, as previously reported (Gavathiotis et al., 2008) and in accordance with standard methods (Marintchev et al., 2007). Residues with significant backbone amide chemical shift change are concentrated in the region of the trigger site (α1, α1-α2 loop, α6) and BH3 domain (α2). BIM SAHB-induced chemical shift of BAX BH3 residue T56, which lies on the inward-facing surface of the BH3 helix in direct contact with α1, becomes even more prominent than previously observed for wild-type BAX(Gavathiotis et al., 2008), in addition to new changes for BH3 residues E61, C62, G67, D68, and N73 of ¹⁵N-BAX(A112C-V177C) and D53, A54, S55, K58, S60, L63, and D68 of ¹⁵N-BAX(P168G). Whereas no α9 chemical shift changes are observed when α9 is covalently tethered, the point mutant construct displays allosteric sensing at the C-terminus in a manner similar to wild-type BAX treated with higher doses of BIM SAHB (Fig. 2A). Cα atoms of affected residues are represented as orange spheres in the ribbon diagram and orange bars in the plot, based on a calculated significance threshold of ≥0.008 p.p.m. and ≥0.015 for chemical shift changes of ¹⁵N-BAX(A112C-V177C) and ¹⁵N-BAX(P168G) (50 μM), respectively, upon addition of BIM SAHB (100 μm). The site of P168G mutagenesis is indicated by a green sphere in the ribbon diagram. (C, D) BIM SAHB triggered dose-responsive exposure of the BAX BH3 domain (residues 53-71) as monitored by immunoprecipitation of BAX(A112C-V117C) (C) and BAX(P168G) (D) using a BAX BH3 antibody and anti-BAX western analysis. See also Supplementary Figure 3.

Figure 4. BAX BH3 propagates BAX activation by engaging the α1/α6 trigger site

(A) Sequence alignment of the BIM and BAX BH3 domains revealed striking amino acid identity of core sequences implicated in BIM SAHB binding to the BAX trigger site. (B) Measured chemical shift changes of ¹⁵N-BAX upon BAX SAHB titration up to a ratio of 1:2 BAX:BAX SAHB are plotted as a function of BAX residue number. Residues with significant backbone amide chemical shift change are concentrated at the trigger site (α1, α1-α2 loop, α6), with notable allosteric sensing again observed, for example, at internal residues T56 of the BH3 domain and T174 of α9. Cα atoms of affected residues are represented as orange spheres in the ribbon diagram and orange bars in the plot, based on a calculated significance threshold of ≥0.014 p.p.m. for chemical shift changes of ¹⁵N-BAX (50 μM) upon addition of BAX SAHB (100 μM). (C, D) BAX SAHB induced BAX oligomerization and BAX-mediated cytochrome c release, an effect that was impaired by K21E mutagenesis of the BAX trigger site. (E, F) E69K mutagenesis of BAX SAHB impaired ligand-induced BAX oligomerization and BAX-mediated cytochrome c release, but restored BAX K21E activity to wild-type levels. (G) The capacity of native BAX BH3 to propagate BAX activation through intermonomeric interaction at the trigger site was explored by complementary mutagenesis. Heat-initiated BAX oligomerization was impaired by K21E mutagenesis of the trigger site or E69K mutagenesis of the BH3 domain, but fully restored by complementary K21E and E69K mutagenesis. Error bars represent the mean +/- s.d. for experiments performed in at least triplicate. See also Supplementary Figure 3.

Figure 5. BH3-triggered structural reorganization drives the BAX activation pathway

Cytosolic BAX activation is initiated by BIM BH3 engagement of the α1/α6 trigger site. A series of discrete structural changes ensue, including α1-α2 loop displacement, 6A7 epitope exposure, BAX BH3 exposure, and α9 release for mitochondrial translocation. BAX propagates its activation through triggering interactions between the exposed BAX BH3 domain of fully activated monomers and the α1/α6 binding site of inactive monomers. BAX assembles into a structurally undefined homo-oligomeric pore that promotes apoptosis by releasing mitochondrial factors such as cytochrome c.