1. A Stapled BID BH3 Helix Directly Binds and Activates BAX
Loren D. Walensky, Kenneth Pitter, Joel Morash, Kyoung Joon Oh, Scott Barbuto, Jill Fisher, Eric Smith, Gregory L. Verdine, Stanley J. Korsmeyer 
Molecular Cell 
Volume 24, Issue 2, Pages (October 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Primary Sequence and α Helicity of Hydrocarbon-Stapled BH3 Peptides
(A) The nonnatural S-pentenyl alanine derivative was synthesized as described (Schafmeister et al., 2000; Williams and Im, 1991) and substituted into the BH3 peptide sequences at the indicated i, i+4 positions. An intramolecular all-hydrocarbon crosslink was subsequently generated by ruthenium-catalyzed olefin metathesis (Blackwell and Grubbs, 1994; Schafmeister et al., 2000). The native methionine of BID BH3 was replaced with norleucine (NL) in BID SAHBA due to the incompatibility of sulfur with the metathesis reaction.
(B) Circular dichroism studies demonstrated that insertion of a hydrocarbon staple into BH3 peptides markedly induced α-helical structure. Compared to their unmodified counterparts, SAHBs demonstrated a 4- to 6-fold increase in α-helical content.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Specific Interaction of BID SAHBA with Multidomain Proapoptotic BAX
(A) Fluorescence polarization binding assays were performed using FITC-labeled peptides (50 nM) and BAX, BAX ΔC, BCL-XL, and BCL-XL ΔC proteins at the indicated concentrations. EC50 values for apoptotic protein binding to FITC-labeled peptides were determined by nonlinear regression analysis of dose-response curves using Prism 4.0 software (GraphPad).
(B) Hydrocarbon stapling revealed a direct interaction between BID SAHBA and full-length BAX. No BAX interaction was observed with BID BH3 or the BID SAHBA(L,D→A) mutant.
(C) Deletion of the BAX C-terminal helix (BAX ΔC) enhanced the BID SAHBA interaction by 3-fold and also enabled binding of the unmodified BID BH3 peptide to BAX, albeit at 3-fold lower affinity than the stapled derivative.
(D and E) BID SAHBA and BID BH3 bound with 5-fold greater affinity to BCL-XL ΔC than full-length BCL-XL.
(F and G) In contrast to BID SAHBA, BAD SAHBA displayed no association with BAX or BAX ΔC. Likewise, no BAX binding was observed for BAD BH3.
(H and I) However, BAD BH3 and BAD SAHBA did engage BCL-XL and BCL-XL ΔC, with an observed ∼2- to 3-fold preference for the C-terminally truncated construct. Error bars reflect the standard deviation from the mean for an experimental condition performed in triplicate.
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4. Figure 3 Selectivity of SAHBA Interactions with BAX In Vivo
(A) To confirm that the mutant BID SAHBA(L,D→A) compound was cell permeable (as previously documented for the parental BID SAHBA), Jurkat T cell leukemia cells were incubated with FITC-BID BH3 and BID SAHBA peptides for 4 hr followed by washing, trypsinization, and FACS analysis to evaluate cellular fluorescence. Whereas BID BH3-treated cells (black) were FITC negative, BID and mutant BID SAHBA-treated cells (blue and green, respectively) both demonstrated cellular fluorescence.
(B) Jurkat T cell leukemia cells were incubated with FITC-BID and mutant BID SAHBAs for 18 hr, followed by cellular lysis in a 1% CHAPS-containing buffer. Anti-FITC pull-down coimmunoprecipitated native BAX with FITC-BID SAHBA, but not with the mutant BID SAHBA(L,D→A).
(C and D) To further confirm the specificity of the in vivo BID SAHBA-BAX interaction, cell permeability and coimmunoprecipitation experiments were repeated using BAD SAHBA. Similar to BID SAHBA, cellular fluorescence was observed in BAD SAHBA-treated (red) but not BAD BH3-treated (pink) Jurkat T cells (C). In contrast to BID SAHBA, BAX was not associated with immunoprecipitated FITC-BAD SAHBA (D).
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5. Figure 4
BID SAHBA Triggers Dose- and Time-Responsive BAX-Induced Mitochondrial Cytochrome c Release
(A) Mitochondria isolated from Bak−/− mice exhibited dose-responsive release of cytochrome c when incubated with BAX (50 nM) and BID SAHBA (50–500 nM). Mitochondrial treatment with BAX or BID SAHBA alone had no such effect. The specificity of BID SAHBA activity was confirmed by the inability of mutant BID SAHBA(L,D→A) or BAD SAHBA to activate BAX-induced cytochrome c release, and by abrogation of BID SAHBA activity upon BCL-XL ΔC cotreatment. The BID SAHBA-triggered release was time dependent (B) and was amplified by cotreatment with BAD SAHBA (C). Error bars reflect the standard deviation from the mean for an experimental condition performed in quadruplicate.
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6. Figure 5
BAK- and BAX/BAK-Deficient Mitochondria Respond Similarly to BID SAHBA-Induced BAX Activation
(A) To confirm that BID SAHBA-triggered mitochondrial cytochrome c release derived from activation of exogenous BAX, liver mitochondria isolated from Bak−/−, Baxflox/floxBak−/−, and Alb-creposBaxflox/−Bak−/− mice were subjected to anti-BAX western analysis. Whereas mitochondrial porin (VDAC) was observed in all preparations, no native BAX was detected in the mitochondrial samples.
(B) We further evaluated whether there was a functional difference between the BID SAHBA-induced BAX effect in Bak−/− mitochondria (Baxflox/floxBak−/−) and those specifically engineered to lack both BAK and BAX (Alb-creposBaxflox/−Bak−/−). We found no statistically significant difference between the responsiveness of the mitochondria to BAX (50 nM) and BID SAHBA (50–200 nM) cotreatment. Error bars reflect the standard deviation from the mean for an experimental condition performed in quadruplicate.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Selective Activation of BAX-Induced Liposomal Release by Nanomolar BID SAHBA and a More Potent Membrane-Targeted Derivative
(A) BID SAHBA (see Figure S3 for the primary sequence of BID SAHBA compounds used in liposomal experiments), but not the corresponding unmodified peptide, induced dose-responsive (50–200 nM) liposomal FITC-dextran release in the presence of BAX (15 nM). BAX or BID SAHBA alone had no effect.
(B) The specificity of BID SAHBA activity was confirmed by the inability of BID SAHBA(L,D→A) mutant or BAD SAHBA to activate BAX. In addition, the BID SAHBA effect was completely blocked by coadministration of excess BCL-XL ΔC.
(C) (His)6-BID SAHBA, targeted to Ni2+-NTA-containing liposomes, demonstrated more potent BAX-induced FITC-dextran release compared to the untargeted compound. The effect of (His)6-BID SAHBA was completely blocked by cotreatment with BCL-XL ΔC and by eliminating BAX from the assay. Liposomal assays were performed in triplicate with similar results.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Enhanced α Helicity and Multidomain Apoptotic Binding Activity of BIM SAHBA
A hydrocarbon-stapled derivative of BIM BH3 (A) was generated as described for BID and BAD SAHBs and exhibited 4-fold enhancement of α helicity compared to the corresponding unstapled peptide (B). BIM SAHBA displayed comparable, high-affinity binding to both full-length BAX and BCL-XL (C). In contrast, BIM BH3 peptide only bound to BCL-XL. Error bars reflect the standard deviation from the mean for an experimental condition performed in triplicate.
Molecular Cell  , DOI: ( /j.molcel )
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