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

Abstract

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 alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

“…In one approach, membrane fractions were incubated with tBid in the presence of the 4B5 antibody that can capture the transiently exposed Bak BH3 domain. 14,24 As expected, 4B5 blocked cytochrome c release mediated by each Bak variant, whereas the control antibody did not ( Figure 5). 4B5 also inhibited linkage at the BH3:groove and α6:α6 interfaces ( Figure 5), as seen previously.…”

“…When cysteines in the BH3:groove and α9:α9 interfaces were combined, higherorder complexes were apparent, and the complexes were similar to those generated by α6:α6 linkage (MK/H164C) (Figure 7a). Thus, as argued for the α6:α6 interface, 24 the α9: α9 interface is distinct from the BH3:groove interface, and can link the BH3:groove dimers (D 1 ) to higher-order oligomers.…”

“…4B5 also inhibited linkage at the BH3:groove and α6:α6 interfaces ( Figure 5), as seen previously. 14,24 However, linkage at α9:α9 was largely unaffected ( Figure 5, top), indicating that the α9 helices can come into proximity after Bak has undergone conformation change but before BH3:groove dimerization.…”

“…Previously, combining linkage at the Bak BH3:groove and α6:α6 interfaces generated higher-order oligomers, indicating the two interfaces are distinct and complementary. 24 We thus generated Bak variants with cysteines positioned at the α9:α9 interface and at interfaces on either side of the MOM: each retained pro-apoptotic function (Supplementary Figure S1g). When cysteines in the BH3:groove and α9:α9 interfaces were combined, higherorder complexes were apparent, and the complexes were similar to those generated by α6:α6 linkage (MK/H164C) (Figure 7a).…”

“…19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.…”