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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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NMR analysis of BAX upon BIM SAHB titrationa, 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 Cα 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.
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Figure 1: NMR analysis of BAX upon BIM SAHB titrationa, 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 Cα 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.

Mentions: BIM SAHB binding to BAX was monitored using Nuclear Magnetic Resonance (NMR) spectroscopy. Compared to the 1H-15N correlation spectrum of BAX, the addition of BIM SAHB broadened and shifted select NMR cross-peaks, indicating fast exchange between the bound and unbound conformations of BAX. The overall features of the NMR spectra are quite similar except for significant changes in the loop residues between α1 and α2 upon BIM SAHB binding. Chemical shift perturbation mapping of BAX with BIM SAHB titration revealed interactions at a discrete subset of BAX residues. The degree of change in the 1H-15N cross-peak positions for backbone amides is shown in Figure 1a. The largest changes were observed for residues localized in the α1 and α6 helices, as well as residues in the flexible loop between α1 and α2. Significant changes were also observed for the side-chain NH2 of Q28, Q32, and Q52 (Supplementary Fig. 2). In the BAX structure35, the α1 and α6 helices are positioned adjacent to one another, and the residues impacted by BIM SAHB binding localize to a discrete site at the juxtaposition of these α-helices on one side of the protein structure (Fig. 1b). Of note, no residues on the carboxy terminal side of the protein are affected by BIM SAHB titration under these conditions, thus placing the novel binding site on the completely opposite face of the protein from the canonical BH3 binding site of anti-apoptotic proteins (Supplementary Fig. 1). The binding site of BIM SAHB on BAX is thus defined by the two helices α1 and α6, with the interhelical junction forming a hydrophobic cleft surrounded by a perimeter of hydrophilic and charged residues (Fig. 1c).


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)

NMR analysis of BAX upon BIM SAHB titrationa, 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 Cα 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: NMR analysis of BAX upon BIM SAHB titrationa, 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 Cα 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.
Mentions: BIM SAHB binding to BAX was monitored using Nuclear Magnetic Resonance (NMR) spectroscopy. Compared to the 1H-15N correlation spectrum of BAX, the addition of BIM SAHB broadened and shifted select NMR cross-peaks, indicating fast exchange between the bound and unbound conformations of BAX. The overall features of the NMR spectra are quite similar except for significant changes in the loop residues between α1 and α2 upon BIM SAHB binding. Chemical shift perturbation mapping of BAX with BIM SAHB titration revealed interactions at a discrete subset of BAX residues. The degree of change in the 1H-15N cross-peak positions for backbone amides is shown in Figure 1a. The largest changes were observed for residues localized in the α1 and α6 helices, as well as residues in the flexible loop between α1 and α2. Significant changes were also observed for the side-chain NH2 of Q28, Q32, and Q52 (Supplementary Fig. 2). In the BAX structure35, the α1 and α6 helices are positioned adjacent to one another, and the residues impacted by BIM SAHB binding localize to a discrete site at the juxtaposition of these α-helices on one side of the protein structure (Fig. 1b). Of note, no residues on the carboxy terminal side of the protein are affected by BIM SAHB titration under these conditions, thus placing the novel binding site on the completely opposite face of the protein from the canonical BH3 binding site of anti-apoptotic proteins (Supplementary Fig. 1). The binding site of BIM SAHB on BAX is thus defined by the two helices α1 and α6, with the interhelical junction forming a hydrophobic cleft surrounded by a perimeter of hydrophilic and charged residues (Fig. 1c).

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
Related in: MedlinePlus