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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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Orientation of BIM SAHB at the novel BAX binding sitea,Paramagnetically-labeled derivatives of BIM SAHB. b, Ratios of BAX crosspeak intensities in the presence of oxidized or reduced BIM SAHBMTSL (Iox/Ired) versus residue number. BAX residue intensities reduced below 0.6 are colored. c, Orientation of BIM SAHB at the BAX site. Based on an Iox/Ired 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.
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Figure 2: Orientation of BIM SAHB at the novel BAX binding sitea,Paramagnetically-labeled derivatives of BIM SAHB. b, Ratios of BAX crosspeak intensities in the presence of oxidized or reduced BIM SAHBMTSL (Iox/Ired) versus residue number. BAX residue intensities reduced below 0.6 are colored. c, Orientation of BIM SAHB at the BAX site. Based on an Iox/Ired 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.

Mentions: One of the challenges of capturing a BAX-activating interaction by NMR is time- and dose-dependent BAX aggregation due to BIM SAHB addition to the NMR sample. With time, the size of the protein aggregate formed leads to line broadening of the NMR resonances beyond detection, thereby precluding acquisitions of data for the duration required to resolve the initial complex by conventional NMR. Nevertheless, to more precisely orient BIM SAHB at the BAX interaction site, we conducted paramagnetic relaxation enhancement (PRE) NMR experiments, which can be performed on a time-scale that is compatible with the lifespan of the complex under NMR conditions. 1H-15N-BAX HSQC spectra were acquired with methane thiosulphonate (MTSL)-derivatized BIM SAHB compounds (Fig. 2a) in the oxidized state and then repeated after nitroxide reduction. Of note, the chemical shift perturbation maps of BAX in complex with BIM SAHB and the MTSL-labeled derivatives demonstrate consistent changes in BAX α-helices 1 and 6, and in the α1-α2 loop (Supplementary Fig. 3). Whereas the intensity of BAX α1 residues are predominantly reduced in the presence of oxidized C-terminal label, the intensity of BAX α6 residues are predominantly decreased by the presence of oxidized N-terminal label (Fig. 2b). As an example, BIM SAHB(A164C-MTSL) caused marked signal suppression of Ser16 (α1) but had essentially no effect on Asp142 (α6) (Supplementary Fig. 4a), whereas BIM SAHB(W147C-MTSL) had no effect on Ser16 (α1) but reduced the Asp142 (α6) signal (Supplementary Fig. 4b). Because the degree of PRE correlates with the distance between the nitroxide label and the affected BAX residues36, structure calculations using restraints derived from PRE and chemical shift perturbation data were performed to define the orientation of BIM SAHB at the novel BAX site. The calculated model structures converged to place BIM SAHB perpendicular to BAX helices α1 and α6, with the N- to C-terminus directionality of the peptide disposed right to left (Fig. 2c, Supplementary Table 1). Whereas the location of the novel BAX binding site is geographically distinct, the calculated model structure of the BIM BH3-BAX complex reveals an interaction topography that is strikingly similar to that of BIM BH3 with anti-apoptotic BCL-XL, MCL-1, and BFL1/A1, as previously determined by X-ray crystallography (Supplementary Fig. 5).


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)

Orientation of BIM SAHB at the novel BAX binding sitea,Paramagnetically-labeled derivatives of BIM SAHB. b, Ratios of BAX crosspeak intensities in the presence of oxidized or reduced BIM SAHBMTSL (Iox/Ired) versus residue number. BAX residue intensities reduced below 0.6 are colored. c, Orientation of BIM SAHB at the BAX site. Based on an Iox/Ired 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.
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Related In: Results  -  Collection

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Figure 2: Orientation of BIM SAHB at the novel BAX binding sitea,Paramagnetically-labeled derivatives of BIM SAHB. b, Ratios of BAX crosspeak intensities in the presence of oxidized or reduced BIM SAHBMTSL (Iox/Ired) versus residue number. BAX residue intensities reduced below 0.6 are colored. c, Orientation of BIM SAHB at the BAX site. Based on an Iox/Ired 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.
Mentions: One of the challenges of capturing a BAX-activating interaction by NMR is time- and dose-dependent BAX aggregation due to BIM SAHB addition to the NMR sample. With time, the size of the protein aggregate formed leads to line broadening of the NMR resonances beyond detection, thereby precluding acquisitions of data for the duration required to resolve the initial complex by conventional NMR. Nevertheless, to more precisely orient BIM SAHB at the BAX interaction site, we conducted paramagnetic relaxation enhancement (PRE) NMR experiments, which can be performed on a time-scale that is compatible with the lifespan of the complex under NMR conditions. 1H-15N-BAX HSQC spectra were acquired with methane thiosulphonate (MTSL)-derivatized BIM SAHB compounds (Fig. 2a) in the oxidized state and then repeated after nitroxide reduction. Of note, the chemical shift perturbation maps of BAX in complex with BIM SAHB and the MTSL-labeled derivatives demonstrate consistent changes in BAX α-helices 1 and 6, and in the α1-α2 loop (Supplementary Fig. 3). Whereas the intensity of BAX α1 residues are predominantly reduced in the presence of oxidized C-terminal label, the intensity of BAX α6 residues are predominantly decreased by the presence of oxidized N-terminal label (Fig. 2b). As an example, BIM SAHB(A164C-MTSL) caused marked signal suppression of Ser16 (α1) but had essentially no effect on Asp142 (α6) (Supplementary Fig. 4a), whereas BIM SAHB(W147C-MTSL) had no effect on Ser16 (α1) but reduced the Asp142 (α6) signal (Supplementary Fig. 4b). Because the degree of PRE correlates with the distance between the nitroxide label and the affected BAX residues36, structure calculations using restraints derived from PRE and chemical shift perturbation data were performed to define the orientation of BIM SAHB at the novel BAX site. The calculated model structures converged to place BIM SAHB perpendicular to BAX helices α1 and α6, with the N- to C-terminus directionality of the peptide disposed right to left (Fig. 2c, Supplementary Table 1). Whereas the location of the novel BAX binding site is geographically distinct, the calculated model structure of the BIM BH3-BAX complex reveals an interaction topography that is strikingly similar to that of BIM BH3 with anti-apoptotic BCL-XL, MCL-1, and BFL1/A1, as previously determined by X-ray crystallography (Supplementary Fig. 5).

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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