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Determinants of BH3 sequence specificity for the disruption of Bcl-xL/cBid complexes in membranes Kushal Kumar Das, Raed Shalaby, and Ana J. García-Sáez ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01084 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017
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Figure 1: Sequence of BH3 peptides and structural insight of the interacting amino acid residues with the groove of Bcl−xL. A) Peptides derived from the BH3 domains of Bid and 7 other canonical human BH3-only proteins (Puma, Bad, Hrk, Bik, Noxa, Bmf, Bim). Sequence alignment shows the hydrophobic residues (h1h4) important for the hydrophobic interaction with the groove of prosurvival proteins. For the measurements in membranes, we purchase the corresponding peptides containing additionally 6X His residues at the Cterminal end, for effective membrane targeting. B) Sequence map showing the conserved amino acids relevant for determining the binding specificity to pro-survival Bcl-2 proteins (blue, green and black represents hydrophilic, neutral and hydrophobic amino acids, respectively). Error bars represent the Bayesian 95% confidence interval. C) Structure of BH3Bim (26 amino acid) interacting with the Bcl-xL hydrophobic groove (Pdb id 4QVF). The gray side chains of the amino acids pointing inside the hydrophobic groove (hydrophobic interactions), the red and blue (charged amino acids) pointing outside the groove (electrostatic interactions) are shown in the zoom-in 69977x60452mm (1 x 1 DPI)
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Figure 2: Binding of human cBidR and Bcl-xLG to giant unilamellar vesicles and quantification of the interaction of human cBidR and Bcl-xLG by fluorescence cross-correlation spectroscopy (FCCS) in solution and in the membrane environment. A) Confocal images of cBidR and Bcl-xLG binding to GUVs. a) Guvs PC: CL (8:2) labeled with 0.05% DiD in red. b) Bcl-xLG binding to the same GUVs, no binding observed. c) GUVs labeled with 0.05% DiO in green. d) cBidR binding to the GUVs in c. e) cBidR binding to GUVs (PC: CL, 8:2). f) cBidR-induced binding of Bcl-xLG to the GUVs in e. Scale bar indicates 5µm B) FCS analysis of the interactions in solution. Fitted AC curves of cBidR and Bcl-xLG are shown in red and green respectively. CC curve is shown in blue. C) FCS analysis of the interactions in the membrane of GUVs. The red, green and blue lines correspond to cBidR, Bcl-xLG AC and CC respectively. D) Diffusion coefficient of cBidR and Bcl-xLG in solution. E) Diffusion coefficient of cBidR and Bcl-xLG in the membrane. F) Quantification of cBidR/Bcl-xLG complexes in solution, expressed as %CC at varying cBidR concentration (molecules/µm3) G) Quantification of cBidR/Bcl-xLG complexes in membrane, expressed as %CC at varying cBidR concentration (molecules/µm2) H) %CC in solution represented in a three dimensional plot as a function of individual protein concentrations. I) %CC in the membrane represented in a three dimensional plot as a function of individual protein concentrations. The error bars represent the SD.
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Figure 3: Quantitative analysis of the inhibitory activity of BH3 peptides in solution using FCCS. A) Schematic representation of the displacement assay. BH3 peptides (blue) disrupt the cBidR/Bcl-xLG complexes by binding to the hydrophobic groove of Bcl-xLG. B) Auto and CC analysis of interactions in solution. The blue curve shows the change in %CC before (high CC) and after the disruption of the complexes (low CC) in solution. C) Quantitative analysis of the disruption of the cBidR /Bcl-xLG complexes in solution by the respective BH3 peptides (non-competitive). The change in the %CC is shown at the different peptide concentrations. The red line represents the maximum CC observed in solution between cBidR and Bcl-xLG when no peptides were present. The error bars represent the standard deviation. D) The normalized change in %CC of the cBidR /Bcl-xLG complexes in solution at different concentrations of the BH3 peptides. The comparative efficienty of the peptides in solution is depicted based on the two-tailed student’s t-test. 89535x62966mm (1 x 1 DPI)
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Figure 4: Quantitative analysis of the inhibitory activity of BH3 peptides in membranes using Scanning FCCS. A) Schematic representation of the displacement assay. cBidR and BH3 peptides (blue) compete for the binding groove in membrane-associated Bcl-xLG. The blue curve shows the change in the %CC before and after the adding the BH3 peptides. B) Representative FCCS graphs of different BH3 peptides. Fitted AC curves of cBidR and Bcl-xLG shown in red and green respectively, CC curve is shown in blue. The line shows the average CC (%) computed after scanning ≥ 20 GUVs. A comparative scheme of the peptides efficiency in membrane environment is also shown, based on the statistical analysis of all peptides (two –tailed students t-test) with respect to Hrk. C) % CC between cBidR and Bcl-xLG in presence of the respective BH3 Peptides. The background crosstalk between two channels red and green is represented in red. The black line represents the mean %CC, and the significance of each peptide with respect to the control (without peptides, representing the maximum possible cross-correlation between cBidR and Bcl-xLG in membranes). Statistically significant differences (Anova Turkey’s multiple comparison test) are indicated. ****P
