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BAX activation is initiated at a novel interaction site.

Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu HC, Kim H, Cheng EH, Tjandra N, Walensky LD - Nature (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

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.

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BIM SAHB directly initiates BAX activation in vitroa, 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- mitochondria upon incubation with BAX and BIM SAHB. Error bars, mean ± s.d.
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Figure 3: BIM SAHB directly initiates BAX activation in vitroa, 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- mitochondria upon incubation with BAX and BIM SAHB. Error bars, mean ± s.d.

Mentions: In contrast to the stable inhibitory interactions of BIM BH3 with anti-apoptotic proteins, the BAX-activating interaction triggers a dynamic continuum of events that includes BAX conformational change and oligomerization (Supplementary Fig. 6). Of particular interest, NMR resonances of residues in the α1-α2 loop of BAX shifted significantly upon BIM SAHB binding (Fig. 1a, Supplementary Fig. 3). In monomeric BAX, the center of the loop weakly associates with residues Ile133 and Met137 of the α6 helix35. Changes observed in the loop residues can readily be explained by the shift of the loop conformation into an open form upon BIM SAHB binding. Because loop displacement upon ligand engagement may initiate a conformational change of BAX, we investigated whether BIM SAHB binding could directly activate BAX in solution. We monitored both the conversion of BAX from monomer to oligomer by size-exclusion chromatography (SEC) and the exposure of its buried N-terminal residues using the 6A7 antibody, which selectively detects the conformational change associated with BAX activation37. Indeed, BIM SAHB triggered dose-dependent oligomerization of BAX (Fig. 3a). This BIM SAHB-induced oligomerization correlated with exposure of the N-terminal epitope of BAX as recognized by the 6A7 antibody (Fig. 3b). Thus, the transient stability of the BIM SAHB-BAX complex we observe by NMR correlates with the interaction-triggered BAX conformational change and oligomerization that we detect biochemically.


BAX activation is initiated at a novel interaction site.

Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu HC, Kim H, Cheng EH, Tjandra N, Walensky LD - Nature (2008)

BIM SAHB directly initiates BAX activation in vitroa, 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- mitochondria upon incubation with BAX and BIM SAHB. Error bars, mean ± s.d.
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Related In: Results  -  Collection

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Figure 3: BIM SAHB directly initiates BAX activation in vitroa, 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- mitochondria upon incubation with BAX and BIM SAHB. Error bars, mean ± s.d.
Mentions: In contrast to the stable inhibitory interactions of BIM BH3 with anti-apoptotic proteins, the BAX-activating interaction triggers a dynamic continuum of events that includes BAX conformational change and oligomerization (Supplementary Fig. 6). Of particular interest, NMR resonances of residues in the α1-α2 loop of BAX shifted significantly upon BIM SAHB binding (Fig. 1a, Supplementary Fig. 3). In monomeric BAX, the center of the loop weakly associates with residues Ile133 and Met137 of the α6 helix35. Changes observed in the loop residues can readily be explained by the shift of the loop conformation into an open form upon BIM SAHB binding. Because loop displacement upon ligand engagement may initiate a conformational change of BAX, we investigated whether BIM SAHB binding could directly activate BAX in solution. We monitored both the conversion of BAX from monomer to oligomer by size-exclusion chromatography (SEC) and the exposure of its buried N-terminal residues using the 6A7 antibody, which selectively detects the conformational change associated with BAX activation37. Indeed, BIM SAHB triggered dose-dependent oligomerization of BAX (Fig. 3a). This BIM SAHB-induced oligomerization correlated with exposure of the N-terminal epitope of BAX as recognized by the 6A7 antibody (Fig. 3b). Thus, the transient stability of the BIM SAHB-BAX complex we observe by NMR correlates with the interaction-triggered BAX conformational change and oligomerization that we detect biochemically.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

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.

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