Figure 4. Pore‐forming activity of Bax proteins with mutations that would reduce the α2–α5 dimer interaction with membranes.

• A
  NMR structure of Bax (α2–α5) dimer and lipid bilayer‐interacting residues. α helices are shown as cylinders. In the α4 and α5 helices, some of the lipid bilayer‐interacting nonpolar, polar, and positively charged residues are shown as orange, green, and blue sticks, respectively, including the R89, F93, F114, A117, and S118 that were mutated to negatively charged residues to disrupt the interaction with lipid headgroups (red ovals) or acyl chains (orange curves). The longest axis (L) of the dimer is tilted 60° from the bilayer normal (N) to maximize the nonpolar interaction with the lipid acyl chains while allowing R89 to interact with the polar lipid headgroups.
• B, C
  Fluorescent dye release from the mitochondria‐mimic liposomes by the indicated wild‐type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye‐specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.
• D
  Digitonin‐permeabilized BMK Bax^−/−/Bak^−/− cells expressing SMAC‐mCherry in the mitochondria intermembrane space (50 μl, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96‐well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC‐mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data were normalized to the percent SMAC‐mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC‐mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data were fit with the [Agonist] vs. response ‐ Variable slope equation␣in GraphPad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.

Source data are available online for this figure.