Limits...
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.

Show MeSH
Sequence specificity of BIM SAHB-induced BAX activationa, BIM SAHBs with differential staple positions and their activities in BAX oligomerization (b) and BAX-mediated cytochrome c release assays (c). d, BIM SAHBA point mutants and their activities in BAX oligomerization (e) and BAX-mediated cytochrome c release assays (f). Error bars, mean ± s.d.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2597110&req=5

Figure 4: Sequence specificity of BIM SAHB-induced BAX activationa, BIM SAHBs with differential staple positions and their activities in BAX oligomerization (b) and BAX-mediated cytochrome c release assays (c). d, BIM SAHBA point mutants and their activities in BAX oligomerization (e) and BAX-mediated cytochrome c release assays (f). Error bars, mean ± s.d.

Mentions: We next performed a “staple scan” and mutagenesis studies to demonstrate and link the specificity of BIM SAHB-induced BAX activation to interaction at the novel site. By performing a staple scan that effectively replaces pairs of amino acid residues within the core BH3 sequence with crosslinked norleucine-like side chains, we simultaneously address which residues are essential to the functional interaction between BIM BH3 and BAX, and further confirm which surface along the BIM BH3 α-helix is essential to BAX engagement (Fig. 4a). Substituting the non-natural amino acid at positions W147, A149, E151, R153, R154, E158, or Y162 did not disrupt BIM SAHB-induced BAX oligomerization (Fig. 4b) or BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a). Indeed, hydrocarbon staples located along the length of the α-helix at surfaces that do not face the hydrophobic contact site on BAX did not disrupt the functional interactions between the BIM SAHB analogs and BAX. However, a staple position that replaced I148 and the highly conserved L152, and localizes to the hydrophobic contact surface for BAX on the BIM BH3 α-helix (Fig. 4a), markedly decreased BAX oligomerization (Fig. 4b) and BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a), findings consistent with the near abrogation of BIM SAHB-induced chemical shift perturbations of 15N-BAX by BIM SAHBC (Supplementary Fig. 8a). In addition, BAD SAHBA, which has the identical staple position as BIM SAHBA but otherwise contains distinct amino acid sequence, did not bind (Supplementary Fig. 8b) or oligomerize (Fig. 4b) BAX, or induce BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a).


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)

Sequence specificity of BIM SAHB-induced BAX activationa, BIM SAHBs with differential staple positions and their activities in BAX oligomerization (b) and BAX-mediated cytochrome c release assays (c). d, BIM SAHBA point mutants and their activities in BAX oligomerization (e) and BAX-mediated cytochrome c release assays (f). Error bars, mean ± s.d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2597110&req=5

Figure 4: Sequence specificity of BIM SAHB-induced BAX activationa, BIM SAHBs with differential staple positions and their activities in BAX oligomerization (b) and BAX-mediated cytochrome c release assays (c). d, BIM SAHBA point mutants and their activities in BAX oligomerization (e) and BAX-mediated cytochrome c release assays (f). Error bars, mean ± s.d.
Mentions: We next performed a “staple scan” and mutagenesis studies to demonstrate and link the specificity of BIM SAHB-induced BAX activation to interaction at the novel site. By performing a staple scan that effectively replaces pairs of amino acid residues within the core BH3 sequence with crosslinked norleucine-like side chains, we simultaneously address which residues are essential to the functional interaction between BIM BH3 and BAX, and further confirm which surface along the BIM BH3 α-helix is essential to BAX engagement (Fig. 4a). Substituting the non-natural amino acid at positions W147, A149, E151, R153, R154, E158, or Y162 did not disrupt BIM SAHB-induced BAX oligomerization (Fig. 4b) or BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a). Indeed, hydrocarbon staples located along the length of the α-helix at surfaces that do not face the hydrophobic contact site on BAX did not disrupt the functional interactions between the BIM SAHB analogs and BAX. However, a staple position that replaced I148 and the highly conserved L152, and localizes to the hydrophobic contact surface for BAX on the BIM BH3 α-helix (Fig. 4a), markedly decreased BAX oligomerization (Fig. 4b) and BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a), findings consistent with the near abrogation of BIM SAHB-induced chemical shift perturbations of 15N-BAX by BIM SAHBC (Supplementary Fig. 8a). In addition, BAD SAHBA, which has the identical staple position as BIM SAHBA but otherwise contains distinct amino acid sequence, did not bind (Supplementary Fig. 8b) or oligomerize (Fig. 4b) BAX, or induce BAX-mediated cytochrome c release (Fig. 4c, Supplementary Fig. 7a).

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.

Show MeSH