Molecular details of Bax activation, oligomerization, and membrane insertion.

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 alpha5 and alpha6) 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.

Topology of the amphipathic helices of the colicin A pore-forming domain in E. coli lipid membranes studied by pulse EPR.

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.

Conformation of the closed channel state of colicin A in proteoliposomes: an umbrella model.

Colicin A (ColA) is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of Escherichia coli. Until now, two models were proposed for the closed channel state: the umbrella model and the penknife model. Mutants of ColA, each containing a single cysteine, were labeled with a nitroxide spin label, reconstituted into liposomes, and studied by electron paramagnetic resonance (EPR) spectroscopy to study the membrane-bound closed channel state. The spin-labeled ColA variants in solution and in liposomes of native E. coli lipid composition were analyzed in terms of the mobility of the nitroxide, its accessibility to paramagnetic reagents, and the polarity of its microenvironment. The EPR data determined for the soluble ColA pore-forming domain are in agreement with its crystal structure. Moreover, the EPR results show that ColA has a conformation in liposomes different from its water-soluble conformation. Residues that belong to helices H8 and H9 are significantly accessible for O(2) but not for nickel-ethylene diamine diacetic acid, indicating their location inside the membrane. In addition, the polarity values determined from the hyperfine tensor component A(zz) of residues 176, 181, and 183 (H9) indicate the location of these residues close to the center of the lipid bilayer, supporting a transmembrane orientation of the hydrophobic hairpin. Furthermore, the accessibility and polarity data suggest that the spin-labeled side chains of the amphipathic helices (H1-H7 and H10) are located at the membrane-water interface. Evidence that the conformation of the closed channel state in artificial liposomes depends on lipid composition is given. The EPR results for ColA reconstituted into liposomes of E. coli lipids support the umbrella model for the closed channel state.