A Stapled BID BH3 Helix Directly Binds and Activates BAX

Abstract

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.

Bax activation by Bim?

“…This places the interaction surface on Bax at a site distinct from the canonical BH3 interaction groove on prosurvival proteins (Figures 1a and 3). We note that fast exchange reported here is indicative of considerably weaker affinity than the value previously reported by these same authors 26 for the stapled BimBH3/ Bax interaction, 24 nM.…”

“…26 In particular, the threefold increase in affinity of the stapled BimBH3 over its parent for Bcl-x L (16 nM compared with 52 nM, respectively, in fluorescence polarization assays) accords with the reported fourfold increase in helicity of the peptide on stapling. Stapled BimBH3 peptides also displayed binding activity for Bax (24 nM) in fluorescence polarization assays but, curiously, no binding to Bax with the unstapled Bim peptide was detected.…”

“…They have synthesized hydrocarbon-stapled BH3 peptides that display enhanced helicity in the absence of a binding partner. 25,26 To construct such peptides, two residues predicted to be on the same face of the helix, are replaced with (S)-pentenyl alanine derivatives and then joined by a metathesis reaction.…”

BH3-only proteins and their roles in programmed cell death

“…While binding of Bid to Bax is relatively easy to observe by coimmunoprecipitation in the presence of non-ionic detergents (Wei et al, 2000;Walensky et al, 2006), binding of Bim to Bax could be shown only for two minor isoforms, BimS and BimAD (Marani et al, 2002), whose physiological significance is also a matter of debate. No direct binding of the major isoform, BimEL, could be observed under the same conditions (Willis et al, 2007), and no binding of any Bim isoform to Bak was ever reported.…”

Measurement of BH3-only protein tolerance

“…15,16 Upon activation, the proapoptotic family members BAK and BAX can breach the mitochondrial outer membrane (MOM), leading to cytochrome c release, caspase 9 activation and subsequent apoptotic events. Genetic 2,17 as well as biochemical experiments [18][19][20][21][22][23] have indicated that the BH3-only proteins BIM, PUMA, a protease generated BID fragment (tBID), and, in some studies, NOXA can directly bind and activate BAK and/or BAX. To counteract these effects, antiapoptotic BCL2 family members such as BCL2, BCLX L and MCL1 bind and neutralize activated BAX and/or BAK 2,6,16,24 as well as activated or overexpressed BH3-only family members, [25][26][27][28][29][30] thereby preserving MOM integrity.…”