Monomer-Monomer Interactions Drive the Prepore to Pore Conversion of a β-Barrel-forming Cholesterol-dependent Cytolysin

Eileen M Hotze,Alejandro P Heuck,Daniel M Czajkowsky,Zhifeng Shao,Arthur E Johnson,Rodney K Tweten

doi:10.1074/jbc.m111039200

Abstract

Perfringolysin O (PFO), a cholesterol-dependent cytolysin, forms large oligomeric pore complexes comprised of up to 50 PFO molecules. In the present studies a mutant of PFO (PFO(Y181A)) has been identified that traps PFO in a multimeric prepore complex that cannot insert its transmembrane beta-hairpins and therefore cannot form a pore. Remarkably, PFO(Y181A) can be induced to insert its transmembrane beta-hairpins if functional PFO is incorporated into the PFO(Y181A) oligomeric prepore complex. Furthermore, the transition from prepore to pore appears to be an "all or none" process; partial insertion of the transmembrane beta-barrel does not occur. Therefore, cooperative interactions between the monomers of the prepore drive the prepore to pore conversion that results in the formation of the transmembrane beta-barrel.

Highlights

Perfringolysin O (PFO), a cholesterol-dependent cytolysin, forms large oligomeric pore complexes comprised of up to 50 PFO molecules
In the present report we describe a novel mutant of PFO, PFOY181A,2 that is trapped in the prepore complex
The emission intensity of the NBD increased significantly in all cases as the ratio of functional PFO (PFOC459A) to PFOY181A increased. These results demonstrated that the transmembrane ␤-hairpins (TMHs) of PFOY181A were inserting into the membrane in the presence of functional PFO, whereas, as shown in Fig. 3, PFOY181A alone cannot insert its TMHs

Summary

Introduction

Perfringolysin O (PFO), a cholesterol-dependent cytolysin, forms large oligomeric pore complexes comprised of up to 50 PFO molecules. Samples of 100 –200 nM NBD-labeled PFOY181A derivatives containing 0 – 400 nM functional PFO (PFOC459A) were incubated with an excess of liposomes (the molar ratio of protein to total lipid was 1:1000) for 40 min at 37 °C before the measurements were made.

Results

Conclusion