Figure 6. h0 and h1 residues in the Bim BH3 contribute to the resistance of Bcl-XL:Bim complexes to ABT-263.

(A) Sequence alignment of the BH3 regions of Bad and Bim and the sequences of ^VBad-Bim mutants, residues from Bad (red), Bim (blue). (B) Identification of conserved hydrophobic residues in the Bim BH3 that contributed to R_ABT-263 for ^CBcl-XL:^VBad-Bim (black) and ^CBcl-2:^VBad-Bim (blue) complexes. The R_ABT-263 values of ^CBcl-XL:^VBad-Bim and ^CBcl-2:^VBad-Bim complexes from Figure 4b and Table 1 were included to facilitate direct comparison. (C) The BH3 h1 residue I146 contributes to Bim binding to Bcl-2 and Bcl-XL. Substitution of I146 with the corresponding residue from Bad (BimEL-I146Y) decreased R_ABT-263 for ^CBcl-XL: ^VBimEL/^VBimEL-dCTS (black) and ^CBcl-2:^VBimEL/^VBimEL-dCTS (blue) complexes. Data are mean ±95% confidence intervals calculated from FLIM-FRET binding curves shown in Figure 6—figure supplement 1.

Figure 6—figure supplement 1. The h0 and h1 residues in the Bim BH3 region contribute to the resistance of Bcl-XL:Bim and Bcl-2:Bim complexes to ABT-263.

(A–L) Binding of ^VBad-Bim chimeric proteins to ^CBcl-XL (left) and ^CBcl-2 (right) in MCF-7 cells. The ^VBad-Bim chimeric proteins are comprised of ^VBad with the BH3 region replaced with the corresponding region from Bim. Additional point mutations in the Bim BH3 region are indicated to the left of each pair of panels. (M–P) Impact of I146 on the binding of ^VBimEL or ^VBimEL-dCTS to ^CBcl-XL (left) and ^CBcl-2 (right) in MCF-7 cells. Additional point mutations in ^VBimEL are indicated to the left of each pair of panels. The binding curves illustrate the extent of binding of untreated (black) and DMSO treated (green) controls, cells treated with 20 μM ABT-263 (blue) or additional BH3-2A mutation (red). Data points in FLIM-FRET binding curves correspond to the average FLIM-FRET efficiency for binned data (20 < cells/bin < 3,000). Error bars, SEM; shadowed area, 95% confidence interval for the best fit of the model to the data.