Fig. 5: BH3 ligand binding to the activation groove regulates helix α1 stability to activate or inactivate BAK.

a BH3 peptide sequences, their binding constants determined from isothermal titration calorimetry (ITC) with BAK, and EC50 values from liposome permeabilization (LP EC50). ITC traces and binding parameters are summarized in Supplementary Fig. 7a. GGC linker is for tethering the peptide to G184C of BAK. b Model for direct BAK activation. c AUC quantification of kinetic traces for liposome permeabilization in Supplementary Fig. 7c. We estimated complex formation according to % bound = ([BID BH3] × 100)/(K_D + [BID BH3]). Data are presented as mean + SEM of n = 2 experiments each of n = 3 technical replicates. Adjusted p values indicated above each bar were calculated by multiple comparisons using one-way ANOVA with Tukey test; ****P < 0.0001. 95% confidence interval of differences are presented in the Source Data File. Cartoon:surface representation for directly activated complex (d) and inactivated complex (f) of BAK bound to BID-like BH3 peptides. Van der Waals contacts (≤4 Å) at the peptide interface are shaded green. The GGC peptide linker is colored blue (d). See also Supplementary Fig. 7 and Supplementary Table 3. e, g Overlay of apo and directly activated and inactivated BAK identifying key amino acids involved in helix α1 stabilization. Apo residues are rendered as sticks and spheres and their electrostatic network contacts (dashed lines) are excluded. Remarkably, the electrostatic network is destabilized and re-stabilized in these complexes, respectively, compared to that observed in apo BAK. Hydrogen bonds ≤3.2 Å (red) and ≤3.6 Å (black) between helix α1 and the rest of the domain identified with * are summarized in brackets, Supplementary Fig. 7g, i, and Supplementary Table 3.