Arresting pore formation of a cholesterol-dependent cytolysin by disulfidetrapping synchronizes the insertion of the transmembrane ss-sheet from a prepore intermediate

Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin fam ily of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced bet ween cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide -trapped mutant (pFO(C190-C57)) was devoid of hemolytic activity and could not insert either of its transmembrane beta -hairpins (TMHs) into the membr ane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys(190)- Cys(57) disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C190-C57 dis ulfide bond, PFOC190-C57 that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO -dependent hemolysis and the membrane insertion of its TMHs when compared w ith toxin that had the disulfide reduced prior mixing the toxin with membra nes. Therefore, the rate-limiting step in pore formation is prepore assembl y, not TMH insertion. These data demonstrate that the prepore is a legitima te intermediate during the insertion of the large transmembrane beta -sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert indepen dently, but instead their insertion is coupled.