Abstract
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 α1 helix are exposed indicating complete dissociation of α1 from α2 in the core and from α6-α8 in the latch. Moreover, disulfide tethering of α1 to α2 or α6 blocks cytochrome c release, suggesting that α1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when α1 is tethered shows that Bid triggers α2 movement, followed by α1 dissociation. However, α2 reaches its final position only after α1 dissociates from the latch. Thus, α1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (α2-α5) and the latch (α6-α8).
Highlights
During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes
To gain insight into the molecular changes occurring at the Bak N terminus during apoptosis, we mapped the epitopes of 11 anti-Bak antibodies using peptide arrays and mutagenesis
We developed a novel fluorescence-activated cell sorting (FACS) protocol for measuring epitope exposure after disulfide tethering, which allowed us to examine the sequence of Bak conformation changes during activation
Summary
Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes We characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. Similar conformation changes occur in Bax during its activation[16,17,18] Another key conformation change identified in Bak and Bax is separation of the latch (a6–a8) from the core (a2–a5)[19,20]. After these large changes in conformation, the proteins form BH3:groove dimers and make significant contact with the outer membrane surface[9,16,19,21,22]. Dissociation of a1 from both the core and latch domains is a key event in the activation of Bak
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