Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices ␣5 and ␣6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.Members of the Bcl-2 protein family are essential players in the complex regulation of apoptosis (1, 2). They are divided into three subgroups: the anti-apoptotic Bcl-2-like proteins, the pro-apoptotic multidomain proteins (Bax and Bak), and the pro-apoptotic BH3 3 -only proteins. To keep programmed cell death under control, Bax activation needs to be strictly regulated, as abnormal cell death is disadvantageous for multicellular organisms.Following cleavage by caspase-8, the BH3-only protein, Bid, is known to activate Bax (3-6). Recently the events involved in BaxBid interaction were investigated by fluorescent techniques (7). Bax is activated through a cascade of conformational changes from being inactive and cytosolic to an oligomeric, membrane-inserted state. In the mitochondrial outer membrane (8, 9) activated Bax is responsible for cytochrome c release and apoptosis initiation (10). Bax oligomerization has been shown to occur also in vitro by incubation with detergents (10 -14).The structures of monomeric Bax and Bid were solved by NMR (14 -16). Bax has a globular fold composed of nine ␣-helices (␣1 to ␣9), with ␣2 representing the BH3 domain and ␣5/␣6 the hydrophobic hairpin (see Fig. 1A). Similarities in structure and function with the monomeric inactive form of the channel-forming domain of bacterial colicins or diphtheria toxins are evident (14, 17). The BH3-only protein, Bid, shows a similar globular fold but lacks ␣8 and ␣9 and has an additional short helix (␣1/2) between ␣1 and ␣2. The structure of active Bax is still unknown, but helices ␣5, ␣6, and ␣9 are reported to in...

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Colicin A is a water-soluble pore-forming protein that kills cells, which are not protected by an immunity protein, by inserting specific helical segments of the toxin subdomain into the cytoplasmic membrane to form voltage-dependent ion channels. This leads to depolarization of the cell membrane followed by depletion of the intracellular ATP levels and finally to cell death. The formation of the integral membrane voltage-gated ion channel is known to be accompanied by a conformational transition. Using double electron electron resonance spectroscopy inter-spin distances in doubly spin labeled colicin A mutants, with spin labels bound to positions 42/187, 62/187, 91/187 and 115/187, have been determined to serve as constraints for the modeling of the membrane bound, closed channel state of colicin A. The data reveal a quasi-circular arrangement of the eight amphipathic helices, embedded in the membrane interfacial layer close to the lipid-water interface, whereas the two hydrophobic helices are buried within the membrane.