Bak Activation for Apoptosis Involves Oligomerization of Dimers via Their α6 Helices

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha 6 helices. Cysteine residues placed in alpha 6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha 6:alpha 6 interface can link these dimers into homo-oligomers, containing at least 18 Bak molecules. A putative zinc-binding site in alpha 6 was not required to form the alpha 6:alpha 6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha 6:alpha 6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.