During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the a1 helix are exposed indicating complete dissociation of a1 from a2 in the core and from a6-a8 in the latch. Moreover, disulfide tethering of a1 to a2 or a6 blocks cytochrome c release, suggesting that a1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when a1 is tethered shows that Bid triggers a2 movement, followed by a1 dissociation. However, a2 reaches its final position only after a1 dissociates from the latch. Thus, a1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (a2-a5) and the latch (a6-a8).

The prosurvival Bcl-2 family proteins Mcl-1 and Bcl-x inhibit apoptosis by sequestering BH3-only proteins such as Bid and Bim (MODE 1) or the effector proteins Bak and Bax (MODE 2). To better understand the contributions of MODE 1 and MODE 2 in blocking cell death, and thus how to bypass resistance to cell death, we examined prescribed mixtures of Bcl-2 family proteins. In a Bim and Bak mixture, Bcl-x and Mcl-1 each sequestered not only Bim but also Bak as it became activated by Bim. In contrast, in a Bid and Bak mixture, Bcl-x preferentially sequestered Bid while Mcl-1 preferentially sequestered Bak. Notably, Bcl-x could sequester Bak in response to the BH3 mimetic ABT-737, despite this molecule targeting Bcl-x. These findings highlight the importance of Bak sequestration in resistance to anti-cancer treatments, including BH3 mimetics.

No abstract