Mutation to Bax beyond the BH3 Domain Disrupts Interactions with Pro-survival Proteins and Promotes Apoptosis

Abstract

The Bcl-2 family of proteins plays a central role in the control of apoptosis via the intrinsic pathway (1). The family is composed of two opposing groups, one promoting cell survival (comprising Bcl-2, Bcl-x L , Bcl-w, Mcl-1, and A1, each containing up to four Bcl-2 homology (BH) 3 domains) and another promoting cell death. This latter group includes Bax and Bak, proteins similar in sequence and structure to the pro-survival family members, and the so-called BH3-only proteins. In response to stress or damage signals, BH3-only proteins are up-regulated either transcriptionally or post-translationally, or both. Apoptosis results when the pro-survival proteins are overwhelmed and Bax/Bak undergo a conformational change at the mitochondrial membrane. Apoptogenic factors such as cytochrome c are then released from the mitochondrial intramembrane space into the cytosol, resulting in the activation of caspases.Many biochemical, structural, and cellular studies support an interaction between pro-survival proteins and both subgroups of pro-apoptotic proteins. The helical BH3 domain of pro-apoptotic proteins engages a cognate groove on the prosurvival proteins (2-5). Four hydrophobic amino acids from the BH3 domain insert into pockets in the groove, and a conserved aspartyl residue in the BH3 domain is hydrogen-bonded with a conserved arginyl residue on the pro-survival protein.Although the BH3-only proteins are mostly unstructured in solution (6), Bak and Bax have well characterized globular structures (7,8), remarkably similar to the pro-survival proteins, in which the BH3 domain is located within an amphipathic helix (␣2) with its hydrophobic face buried. Thus Bak and Bax cannot bind to pro-survival proteins without first everting their BH3 domains. The natural trigger for the exposure of the BH3 domain of Bak/Bax is not known, but it could be a chemical stimulus such as H 2 O 2 (9), a physical stimulus such as heat (10), or an interaction between Bak/Bax and a BH3-only protein that is up-regulated in response to cellular stresses. Some evidence supports such interactions, at least between a subset of the BH3-only proteins and Bax/Bak (11-15). The isolation of complexes of this type has proven elusive, but a recent NMR study suggests the BH3 domain of the BH3-only protein Bim binds Bax on the opposite face as compared with where a BH3 peptide binds a pro-survival protein (16). It has been proposed that this interaction triggers a structural rearrangement in Bax or Bak into large homo-oligomeric complexes at the mitochondrial membrane. Other data support a model in

Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis

“…Interestingly, the A179L E76:ssBid R81 ionic interaction is also found in the variola virus F1L:Bid complex in the form of F1L E143:Bid R83 (20), unlike the Bcl-x L , Mcl-1, A1, and Bcl-w complexes with Bid, where this interaction is absent (37,45,47,48). In contrast, the two additional ionic interactions present in the A179L:Bax BH3 complex formed by D80:K64 and K79:E61 are also conserved in the Bcl-2:Bax BH3 complex as D140:R64 and R139:E61 (36), whereas in the poxviral DPV022:Bax BH3 complex (26) and Mcl-1:Bax complex they are absent (49). In Bcl-x L :Bax BH3, only one of the two ionic interactions is present in the form of Bax K64 with Bcl-x L E129 and D133 (49).…”

Antiapoptotic potency of Bcl-2 proteins primarily relies on their stability, not binding selectivity

“…20,21 Also, removal of the membrane anchor from the prosurvival Bcl-2 proteins can alter binding properties, [22][23][24] and BH3-peptides may not represent the full-length proteins. 25,26 Most likely for these reasons, BH3-binding profiles of Bak and Bax determined in vitro differ greatly from those determined by coimmunoprecipitation from cells 27 ( Figure 2). However, immunoprecipitation studies are not fully consistent either (Figure 2 lines 2-3), because the detergents required to solubilize the mitochondrial membrane can induce or disrupt Bcl-2 family interactions 28,29 or alter Bcl-2 protein conformation, as shown for Bcl-xL, 30 Bcl-w, 31 and Bax.…”

Bcl2L12 with a BH3-like domain in regulating apoptosis and TMZ-induced autophagy: A prospective combination of ABT-737 and TMZ for treating glioma

“…Our finding of h1, h2 and h4 as important residues in the core motif (position -4, 0, and +7) of BH3-like domain of Bcl2L12 may reflect that they serve to interact with Bcl-xL and Bcl2. Interestingly, mutations on corresponding residues of Bax also disrupted the interaction with Bcl2 pro-survival members leading to failure in inducing apoptosis (30).…”

“…The specific primers containing desired mutations [h1, h2, h3, charge and h4, partly as designated as previous report (30)] and the complementary sequences surrounding to mutation sites were designed and synthesized. After PCR amplification, 1.5 µl DpnI (10 U/ µl) was added for digestion at 37˚C for 10 min.…”