Arresting Pore Formation of a Cholesterol-dependent Cytolysin by Disulfide Trapping Synchronizes the Insertion of the Transmembrane β-Sheet from a Prepore Intermediate

Eileen M Hotze,Elizabeth M Wilson-Kubalek,Jamie Rossjohn,Michael W Parker,Arthur E Johnson,Rodney K Tweten

doi:10.1074/jbc.m009865200

Abstract

Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between 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 membrane 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 disulfide bond. PFO(C190-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 with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane beta-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.

Highlights

Perfringolysin O (PFO), a member of the cholesteroldependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers
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 transmembrane ␤-hairpins (TMHs) when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes
Even though PFOC190-C57 was hemolytically inactive before reduction of the disulfide, it was clear that the cytolytic mechanism had progressed to a stage that largely eliminated the typical lag period

Summary

EXPERIMENTAL PROCEDURES

Design of the Disulfide in PFO—Potential sites for cysteine residue pairs that might form disulfide bonds were selected using the method of Hazes and Dijkstra [15]. Labeling of the cysteine derivatives, PFOC190-C57/C215, PFOC190-C57/C202, PFOC190-C57/C288, and PFOC190-C57/C303, with IANBD or IATR was performed as previously described [7, 8], except that 3 M guanidine hydrochloride and DTT were not used in the labeling procedure. Emission scans of NBD-labeled residues in TMH1 (Cys215 or Cys202) or TMH2 (Cys288 or Cys303) were recorded in both the presence and absence of 10 mM DTT in 2 ml of buffer A (50 mM HEPES (pH 7.5), 100 mM NaCl) at 37 °C. Excess liposomes were added to monomeric NBDlabeled PFOC190-C57 derivatives (185 nM) and allowed to incubate for 30 min at 37 °C to ensure that oligomerization and insertion into the membrane were complete before the intensity measurements were made.

Hemolytic activity

RESULTS

DISCUSSION