Bax, despite being a cytosolic protein, has the distinct ability to form channels in the mitochondrial outer membrane, which are capable of releasing proteins that initiate the execution phase of apoptosis. When studied in a planar phospholipid membrane system, full-length activated Bax can form conducting entities consistent with linearly organized three-channel units displaying steep voltage-gating (n=14) that rivals that of channels in excitable membranes. In addition, the channels display strong positive co-operativity possibly arising from the charge distribution of the voltage sensors. On the basis of functional behaviour, one of the channels in this functional triplet is oriented in the opposite direction to the others often resulting in conflicts between the effects of the electric field and the positive co-operativity of adjacent channels. The closure of the first channel occurs at positive potentials and this permits the second to close, but at negative potentials. The closure of the second channel in turn permits closure of the third, but at positive potentials. Positive co-operativity manifests itself in a number of ways including the second and the third channels opening virtually simultaneously. This extraordinary behaviour must have important, although as yet undefined, physiological roles.

It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.

The interaction of two Bacillus thuringiensis cytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the α-helical N-terminal half and β-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly β-strands was inserted into the membrane, whereas the N-terminal half comprising mainly α-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major β-strands (β-5, β-6 and β-7) in the C-terminal half of the toxin.