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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. SYNOPSIS: While BAK is a known executioner protein in apoptotic cell death, how it changes conformation to form oligomeric mitochondrial pores to kill cells remains unclear. Hydrogen‐deuterium exchange mass spectrometry and site‐directed mutagenesis now resolve key events in BAK activation as it occurs on membranes and mitochondria. Hydrogen‐deuterium exchange mass spectrometry provides new insight into conformation changes of BAK on a mitochondria‐like membrane.The BAK BH4 domain constrains BAK activity in the absence of a death stimulus.During BAK activation and oligomerisation, amino acids preceding the BH3 domain become exposed and disordered.The disordered N‐terminus is not required for BAK to permeabilise membranes and mediate cytochrome c release and may rather inhibit it. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co‐ordinate the removal of damaged mitochondria. We report that following mitochondrial damage‐induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3‐only proteins and its homo‐dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX‐mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK‐dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1‐dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro‐inflammatory effect. Synopsis: The ubiquitin ligase Parkin plays a protective role in neurodegenerative disease by removing damaged mitochondria and preventing apoptosis, and by limiting the functions of pro‐apoptotic effector proteins BAK and BAX. Defective control of apoptosis may contribute to the pathogenesis of early onset Parkinson's Disease caused by Parkin mutations. Parkin activity is induced by mitochondrial damage during apoptosis.Parkin mono‐ and di‐ubiquitinates BAK at a conserved lysine in its hydrophobic groove.Ubiquitination reduces BAK oligomerisation and apoptotic activity on mitochondria.Parkin prevents BAX mitochondrial localisation and apoptotic activity independent of ubiquitination.Certain Parkinson's Disease‐associated Parkin mutants cannot ubiquitinate BAK and restrain it on mitochondria. Autophagic clearance of damaged mitochondria and suppression of apoptotic effector proteins are coordinated functions of the Parkin ubiquitin ligase that are lost upon Parkinson's Disease‐associated mutations. [ABSTRACT FROM AUTHOR]