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...

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