Dynamic reconfiguration of pro‐apoptotic BAK on membranes

Abstract

BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self‐associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane‐bound BAK, we employed hydrogen‐deuterium exchange mass spectrometry (HDX‐MS). The HDX‐MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX‐MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX‐MS profiling revealed that the BCL‐2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N‐terminal region preceding the BAK oligomerisation domains became disordered post‐activation and remained disordered in the activated oligomer. Removal of the disordered N‐terminus did not impair, but rather slightly potentiated, BAK‐mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX‐MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.

Structural basis of BAK activation in mitochondrial apoptosis initiation

“…7 inset and Supplementary Movie 1 ). NMR and hydrogen-deuterium exchange mass spectrometry experiments showed that residues 1–65 are intrinsically disordered in BH3 ligand-activated BAK 31 , 32 . Corroborating this mechanism, the T m of activated BH3-BAK complexes is lower than that of apo BAK, and autoactivation proceeds at lower doses of protein with mutants that disrupt the electrostatic network stabilizing helix α1 in apo BAK.…”

Bcl-2 Family Members and the Mitochondrial Import Machineries: The Roads to Death

“…A recent study by HDX-MS showed that the activation of liposomes-bound BakΔC by cBid was associated with an increase in the disorder of the N-terminal part of the protein, leading to a decrease in potential interactions between helix α1 and helices α6–α8 [ 78 ]. This suggests that the N-terminal part of Bak has somehow a negative regulatory function on the activation process.…”

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

“…In an irreversible step toward cell death, BAK and BAX promote apoptosis via mitochondrial outer membrane permeabilization (MOMP) in response to a variety of signals (Tait & Green, 2010). Structural, biochemical, and cell biological studies have illuminated some aspects of the mechanism of membrane permeabilization by BAK and BAX, yet a full understanding remains elusive due to the dynamic nature of these proteins, the lipid environment in which they function, and the complex multilayered BCL-2 network that provides regulatory control (Sandow et al, 2021) (Delbridge et al, 2016). In this study, we addressed early steps in the activation of BAK in which the binding of certain BCL-2 family members to form heterodimers leads to a conformation change that is required for downstream steps including BAK dimerization and higher-order oligomerization.…”