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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.

SUBMITTER: Sandow JJ

PROVIDER: S-EPMC8521275 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Dynamic reconfiguration of pro-apoptotic BAK on membranes.

Sandow Jarrod J JJ Tan Iris Kl IK Huang Alan S AS Masaldan Shashank S Bernardini Jonathan P JP Wardak Ahmad Z AZ Birkinshaw Richard W RW Ninnis Robert L RL Liu Ziyan Z Dalseno Destiny D Lio Daisy D Infusini Giuseppi G Czabotar Peter E PE Webb Andrew I AI Dewson Grant G

The EMBO journal 20210915 20

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 w ...[more]

PMID: 34523147

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Project description:Apoptosis acts in defense against microbial infection, and many infectious agents have developed strategies to inhibit host cell apoptosis. The human pathogen Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that strongly inhibits mitochondrial apoptosis of its human host cell but there is no agreement how the bacteria achieve this. We here provide a molecular analysis of chlamydial apoptosis-inhibition in infected human cells and demonstrate that the block of apoptosis occurs during the activation of the effectors of mitochondrial apoptosis, Bak and Bax. We use small-molecule Bcl-2-family inhibitors and gene targeting to show that previous models cannot explain the anti-apoptotic effect of chlamydial infection. Although the anti-apoptotic Bcl-2-family protein Mcl-1 was strongly upregulated upon infection, Mcl-1-deficient cells and cells where Mcl-1 was pharmacologically inactivated were still protected. Ctr-infection could inhibit both Bax- and Bak-induced apoptosis. Apoptotic Bax-oligomerization and association with the outer mitochondrial membrane was reduced upon chlamydial infection. Infection further inhibited apoptosis induced conformational changes of Bak, as evidenced by changes to protease sensitivity, oligomerization and release from the mitochondrial porin VDAC2. Mitochondria isolated from Ctr-infected cells were protected against the pro-apoptotic Bcl-2-family proteins Bim and tBid but this protection was lost upon protease digestion. However, the protective effect of Ctr-infection was reduced in cells lacking the Bax/Bak-regulator VDAC2. We further found that OmpA, a porin of the outer membrane of Ctr, associated upon experimental expression with mitochondria and inhibited apoptosis, phenocopying the effect of the infection. These results identify a novel way of apoptosis inhibition, involving only the most downstream modulator of mitochondrial apoptosis and suggest that Chlamydia has a protein dedicated to the inhibition of apoptosis to secure its survival in human cells.

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Dynamic reconfiguration of pro-apoptotic BAK on membranes.

Publications

Dynamic reconfiguration of pro-apoptotic BAK on membranes.

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