Movement of a Loop in Domain 3 of Aerolysin Is Required for Channel Formation

Abstract

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

Channels formed by subnanomolar concentrations of the toxin aerolysin trigger apoptosis of T lymphomas

“…This appears to rule out the possibility that apoptosis induced by native toxin resulted from clustering of GPI-anchored receptors such as Thy-1, which might accompany toxin oligomerization. Instead, the results suggest that cell death is caused by channel formation, as T253C/A300C is totally inactive (Rossjohn et al, 1998) and Y221G is nearly inactive. The fact that proaerolysin-induced apoptosis does not result from an interaction with GPI-anchored receptors was con®rmed by experiments with EL4 (Thy-1-f) cells, which do not display GPI-anchored receptors (Fatemi and Tartakoff, 1988).…”

“…The failure of the T253C/A300C variant to kill the cells is also evidence that aerolysin never reaches a reducing environment inside the cell. This variant becomes active when the disulphide bond between 253C and 300C is reduced (Rossjohn et al, 1998), and it might be expected that its uptake and exposure to the cytoplasm would be lethal.…”

“…Rat erythrocytes were incubated with 5´10 À8 , 5´10 À9 or 5´10 À10 M wild-type aerolysin (lanes 1±3 respectively) or aerolysin variant Y221G (lanes 4± 6). The erythrocyte membranes were then isolated and separated by SDS±PAGE and blotted as described previously (Rossjohn et al, 1998). The positions of aerolysin monomer (A) and oligomer (O) are indicated.…”

“…Proaerolysin is an inactive precursor of aerolysin that can be converted to the active toxin by proteolytic nicking after it has bound to the cell (Parker et al, 1996). The T253C/A300C variant of proaerolysin can bind normally, and it is correctly processed, but it is unable to oligomerize and is totally inactive (Rossjohn et al, 1998). The Y221G variant can bind and oligomerize normally (Fig.…”

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

“…Some of them have been shown to cause lysis by opening pores, or forming channels in the cell membrane. Cecropins (6), δ-toxin from Staphylococcus aureus (7), Actinobacillus pleuropneumoniae exotoxins (8), Bordetella pertussis adenylate cyclase toxin (9), Vibrio cholerae hemolysin (10), Escherichia coli α-hemolysin (11), Proteus vulgaris and Morganella morganii hemolysins (12), aerolysin from Aeromonas hydrophila (13,14), gramicidin A from Bacillus brevis and alamethicin from Trichoderma viride (15), melittin (16,17), and sticholysin from the sea anemone Stichodactyla helianthus (18) have all induced channel formation in lipid membranes.…”