BCL-2 and BOK Regulate Apoptosis by Interaction of Their C-terminal Transmembrane Domains

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target. The Bcl-2 family proteins BCL-2 and BOK interact via their transmembrane domains (TMDs) at the endoplasmic reticulum (ER). The TMD interaction interface is critical for the inhibition of BOK-induced apoptosis.BOK and BCL-2 directly interact via their C-terminal transmembrane domains in the ER membrane.Molecular-dynamics simulations reveal formation of higher order oligomers and mutation studies verify the relevance of predicted key residues in the BCL-2-TMD and BOK-TMD interaction interface.Inhibition of BOK-induced cell death by BCL-2 depends on TMD interaction thus exemplifying functional impact of TMD interaction on apoptosis regulation. The Bcl-2 family proteins BCL-2 and BOK interact via their transmembrane domains (TMDs) at the endoplasmic reticulum. The TMD interaction interface is critical for the inhibition of BOK-induced apoptosis.