To Trigger Apoptosis, Bak Exposes Its BH3 Domain and Homodimerizes via BH3:Groove Interactions

Abstract

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

BH3-only proteins and their roles in programmed cell death

“…The list of possible activator BH3-only proteins may not be complete as yet, and Puma is sometimes considered as one (Cartron et al, 2004). Why the Puma BH3 peptide did not prove to be an activator Dewson et al (2008) and Green and Chipuk (2008), we speculate that the binding of stapled Bim BH3 peptide might expose Bax BH3. Such activated Bax molecules could either be captured and inactivated by free pro-survival proteins (represented here by Bcl-2), or, if the pro-survival proteins are saturated with BH3-only proteins, homo-dimerize as a first step toward the formation of 'pores' in the outer mitochondrial membrane.…”

“…In healthy cells, Bax appears to be a monomeric protein. A recent paper also proposes that almost all Bak would also be in the non-activated conformation in healthy cells (Dewson et al, 2008). This is actually not in conflict with the indirect model, because the activation of Bax and Bak could occur soon after the apoptotic stimulus, and the activated proteins could then be captured by pro-survival proteins when they expose their BH3 domain.…”

“…Pore formation involves altered conformation and oligomerization for both Bax and Bak (Eskes et al, 2000;Wei et al, 2000). Recent results about Bak suggest that, upon receiving an apoptotic stimulus, the inactive Bak molecules present on the mitochondrial membrane become activated and expose their BH3 domain (Dewson et al, 2008). This allows dimerization of these activated forms by the insertion of the BH3 domain of one Bak molecule into the hydrophobic groove of another.…”

“…This allows dimerization of these activated forms by the insertion of the BH3 domain of one Bak molecule into the hydrophobic groove of another. This BH3:groove interaction is postulated to produce symmetric Bak dimers that presumably contain additional interaction interfaces to produce higher order oligomers, resulting in homo-oligomerization and permeabilization of the outer mitochondrial membrane (Dewson et al, 2008). Bax is also suggested to undergo similar steps of activation and oligomerization.…”

“…It is noteworthy that none of these major rearrangements were visible in the chemical shifts associated with the binding of the Bim peptide to Bax. An accompanying News and Views article (Green and Chipuk, 2008) drew a parallel with the recently described activation of Bak (Dewson et al, 2008) and speculated that binding of Bim to Bax might ultimately expose the Bax's BH3 domain and allow 'nose-to-nose' dimerization of two activated Bax molecules. This is an interesting possibility indeed, to which we would like to add another: the activated Bax molecule could also be captured by any free pro-survival protein, until such time when free pro-survival proteins are rendered unavailable by the accumulation of BH3-only proteins (Figure 3).…”