Interactions among Bcl-2 family proteins mediated by Bcl-2 homology (BH) regions transform apoptosis signals into actions.The interactions between BH3 region-only proteins and multi-BH region proteins such as Bax and Bcl-2 have been proposed to be the dominant interactions required for initiating apoptosis. Experimental evidence also suggests that both homo-and hetero-interactions are mediated primarily by the BH3 regions in all Bcl-2 family proteins and contribute to commitment to or inhibition of apoptosis. We found that a peptide containing the BH3 helix of Bax was not sufficient to activate recombinant Bax to permeabilize mitochondria. However, an extended peptide containing the BH3 helix and additional downstream sequences activated Bax to permeabilize mitochondria and liposomes. Bcl-2 inhibited the membrane-permeabilizing activity of peptide-activated Bax. This activity of Bcl-2 was inhibited by the extended but not the BH3-only peptide despite both peptides binding to Bcl-2 with similar affinity. Further, membrane-bound Bax activation intermediates directly activated soluble Bax further permeabilizing the membrane. Bcl-2 inhibited Bax auto-activation. We therefore propose that Bax auto-activation amplifies the initial death signal produced by BH3-only proteins and that Bcl-2 functions as an inhibitor of Bax auto-activation.The dominant form of programmed cell death, apoptosis, plays an essential role in embryogenesis and tissue homeostasis of multicellular organisms by removing unwanted or damaged cells. Impaired regulation of apoptosis is implicated in a variety of diseases including cancer and stroke. Two kinds of signals can trigger apoptosis, death signals received by death receptors on cell surface, and stress signals such as structural and genotypic damage. The latter apoptotic signals mainly route through mitochondria to provoke activation of caspases and nucleases that cleave critical proteins and DNAs thereby dismantling the cell (1, 2).Apoptosis is regulated by a complicated series of interactions between Bcl-2 family proteins. These interactions include hetero-and homo-interactions of proteins containing either multiple Bcl-2 homology (BH) 3 regions (BH1-3 or BH1-4) or a single (BH3) region. After receiving certain apoptotic stimuli, pro-apoptotic BH3-only proteins induce the release of cytochrome c and other pro-apoptotic proteins from mitochondria by triggering permeabilization of the mitochondrial outer membrane (MOM) by the multi-BH domain proteins Bax and Bak (3, 4). One proposed mechanism of MOM permeabilization involves BH3-only protein-induced conformational changes in Bax and/or Bak leading to their oligomerization in the MOM (5-7).BH3-only proteins are found in a variety of different subcellular locations and share very limited sequence similarity, often only in the short BH3 region. There is insufficient sequence similarity in BH3 region sequences to permit unambiguous identification from sequence alone. It appears that BH3 region proteins act as sensors within the cell, reporting on...

Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the a5 helix was released into the MOM, the remaining interface with a2, a3, and a4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel a9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, a9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by a9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by a9 dimerization at the rim.

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1-3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1-3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1-3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.

The homo-and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/ Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.Bcl-2 family proteins are key regulators of apoptosis. These proteins share sequence homology in Bcl-2 homology (BH) 1 domains and function to promote or prevent apoptosis. Antiapoptotic proteins such as Bcl-2 and Bcl-x L show homology in four BH domains (BH1 to -4). Proapoptotic proteins can be grouped into "multidomain" and "BH3-only" subfamilies. Multidomain proapoptotic proteins such as Bax and Bak display homology in BH domains 1-3, whereas BH3-only proteins such as Bid and Bim are similar structurally to multidomain family members, but sequence similarity is limited to only the BH3 domain. The current model for how Bcl-2 family proteins regulate apoptosis involves three sequential processes: (i) BH3-only proteins are activated by various death signals; (ii) the active BH3-only proteins then either activate multidomain proapoptotic proteins or inhibit antiapoptotic proteins or both; and (iii) unless inhibited by antiapoptotic Bcl-2 proteins, activated multidomain proapoptotic proteins form oligomers in the mitochondrial outer membrane that release proapoptotic proteins such as cytochrome c and Smac/DIABLO from the mitochondrial intermembrane space. The released proteins trigger activation of the caspases and nucleases that eventually dismantle the cell (1-4).

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a singlespanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.Proteins of the Bcl-2 family are key regulators of apoptosis that function either as promoters or inhibitors and that display homology in one to four short sequences termed Bcl-2 homology (BH) 3 motifs (1-3). Anti-apoptotic subfamily proteins such as Bcl-2 and Bcl-x L contain four BH motifs (BH1-4). Pro-apoptotic proteins are grouped into either multiple or single BH motif subfamilies. The former subfamily includes the proteins Bax and Bak that display homology in BH1-3 motifs. The latter includes proteins such as Bid, Bim, and Bad that are similar only in the limited BH3 motif. Despite the limited sequence homology, the three-dimensional structures determined for seven Bcl-2 family proteins including members from all three subfamilies are very similar, consisting of a hydrophobic core of one to three helices wrapped by five to eight amphipathic helices and their connecting loops. This structure is strikingly similar to the structure of the pore-forming domains of diphtheria toxin and Escherichia coli colicins. In addition there is an obvious hydrophobic groove on the surface of all of the anti-apoptotic Bcl-2 family proteins (4 -6). From decades of extensive studies, two properties are well known to be shared by most, if not all, Bcl-2 family proteins: homo-or hetero-binding and pore formation in membranes. More experiments have been done on the homo-or heterobinding of Bcl-2 family p...

Both proapoptotic Bax and antiapoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.

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