Abstract 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 affects 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 so far very peculiar pro-apoptotic Bcl-2 protein. Interaction of BOK-TMD with BCL-2-TMD localizes at the endoplasmic reticulum (ER). Molecular dynamics simulations in an ER membrane model confirm dynamic BOK-TMD and BCL-2-TMD homo- and heterodimers and stable heterotetramers. 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.

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains