Playing FullBAK

BAK is a pro-apoptotic BCL-2 protein that forms toxic mitochondrial pores in response to cellular stress. A membrane-embedded protein historically refractory to recombinant expression in full-length form, BAK plays it close to the vest. We recently produced monomeric, full-length BAK (FL-BAK) for the first time, 1 providing new opportunities to interrogate its activation mechanism as compared with its close homolog BAX. Unlike BAX, soluble FL-BAK auto-translocates to isolated mitochondria, preferring the membrane environment, consistent with the relative subcellular distributions of BAX and BAK in vivo. FL-BAK translocation did not permeabilize the mitochondria unless a triggering ligand was present. Indeed, we measured direct, sequence-dependent interactions between FL-BAK and an activating BH3 helix. The BH3-only protein tBID, or a hydrocarbon-stapled version of its BH3 helix, oligomerized FL-BAK in the absence of any other factors, definitively demonstrating a direct activation mechanism. 1 Applying photoreactive BH3 helices and MS analysis, we mapped the trigger site for direct BAK activation to its canonical pocket at the C-terminal face, whereas the same ligands crosslinked to BAX at the novel N-terminal trigger site and, upon allosteric release of its C-terminal helix, to the exposed canonical pocket as well. 1 Our analyses suggest that activating BH3 interactions at the canoni-cal pocket in the context of the mitochon-drial membrane may represent a common mechanism for propelling the activation and oligomerization of BAK/BAX, with the N-terminal triggering mechanism of cytosolic BAX representing a unique afferent step required to initiate its activation and mitochondrial translocation. With new rules of the game continually revealed, the competitive interplay among BCL-2 family members has made for an exciting spectator sport that pits cellular life against cellular death. BCL-2 proteins regulate apopto-sis through complex and strategic plays among team members. On offense are BAK and BAX, two formidable teammates that, when activated by BH3 stress signal-ing, spring into action and score, forming a game-ending victory circle at the mito-chondrial outer membrane—the end zone of the apoptotic pathway. The defense is comprised of nimble players like BCL-2 and MCL-1 that block the activated forms of BAK/BAX, stopping the death clock to keep the game alive. In large part, which team wins depends on who is carrying the ball, represented by the critical BH3-only proteins, an elite subgroup of pro-apoptotic BCL-2 family players whose only weapon for interaction is their single BH3 helix. Fortunately, during stressful circumstances, the quarterback has an arsenal of diverse BH3s to launch, …