Bax dimerizes via a symmetric BH3:groove interface during apoptosis

Abstract

During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the a6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax a6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.

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

“…For example, the α2-α5 core dimers of Bak (and of Bax) form a tight helical bundle in X-ray structures, 6,7 confirmed by~100% linkage at the BH3:groove interfaces in mitochondria experiments (e.g., Figure 3a). 14,18 Those core dimers lie in-plane on the MOM surface, as suggested by hydrophobic residues on the bent planar surface of the structures, 6,7 and more recently by IASD labeling of oligomeric Bak and oligomeric Bax in mitochondria experiments. 23 The α6-helix also lies in-plane, however, in contrast to IASD labeling of the BH3:groove interface, IASD could label all tested α6 residues except for those embedded in the MOM, 23 indicating that α6 does not engage in tight protein-protein interactions.…”

“…Linkage at Bak α9:α9 could link BH3:groove dimers to the higher-order oligomers that are associated with pore formation. 26,27 The dimers can also be linked via cysteine residues placed in α6, 18,24,25 and more recently in α3 or α5, 6,21 suggesting a variety of dimer arrangements in the high-order complexes.…”

“…Cytosol and membrane fractions were separated by centrifugation at 13 000 × g for 5 min, and linkage performed as for BaxS184L. 18 …”

“…6,7,[14][15][16] The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 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.…”

“…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.…”