Structural basis of BAK activation in mitochondrial apoptosis initiation

Abstract

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use a similar binding mode and are not distinguished by binding affinity or kinetics

“…We also compared BAK-bound activator dM2 with crystal structures of core-latch domain-swapped dimers of BAK bound to BAK BH3 (PDB: 7M5C) and BIM-RT (PDB:5VWV), which are both activating peptides (G. Singh et al, 2022).…”

“…Given the previously noted importance of residues at hydrophobic positions, we carefully examined the hydrophobic residues that engage the hydrophobic pockets of BAK and found that, in agreement with the similar binding modes, all activators and inhibitors aligned well at the hydrophobic positions ( Figure S18B ). Singh et al have reported the presence of an electrostatic network that includes α1 helix and involves residues R42, E46, D90, N86, Y89, R137 in BAK (G. Singh et al, 2022). We found that the BAK:BAK BH3 complex shows differences in this network compared to the 5 other BAK complexes ( Figure S18B ).…”

“…Other groups have reported the presence of a cavity at the protein-peptide interface of complexes of BAK and BAX bound to BH3 peptides, and it has been suggested that this is important for BH3-induced destabilization and activation (Czabotar et al, 2013) (Brouwer et al, 2017) (G. Singh et al, 2022). The BAK:BIM-RT peptide complex cavity is located between BAK α1, α2, α3, and α5 helices with an estimated volume of 435 Å 3 , while the BAX:BID peptide complex is located between BAX helices α2, α5, and α8 with an estimated volume of 140 Å 3 (Brouwer et al, 2017) (Czabotar et al, 2013).…”

“…For example, BNIP5 is an activator and tight binder, with an affinity of 424 nM. A more recent study also shows that tight-binding peptides M(3)W(5) BID BH3 (Kd = 690 nM) and M(3)W(5) BID BH3 (Kd = 250 nM) can activate BAK in liposomes at 1 µM (G. Singh et al, 2022). Leshchiner et al have also shown that stapled BID SAHB peptides with low nanomolar affinities for binding to murine full-length BAK activate in liposome-based assays (Leshchiner et al, 2013).…”

“…For example, BNIP5 is an activator and tight binder, with an affinity of 424 nM. A more recent study also shows that tight-binding peptides M(3)W(5) BID BH3 (K d = 690 nM) and M(3)W(5) BID BH3 (K d = 250 nM) can activate BAK in liposomes at 1 μM (G. Singh et al, 2022).…”