Structure of a Cholesterol-Binding, Thiol-Activated Cytolysin and a Model of Its Membrane Form

Abstract

The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

“…Diffraction data from three-dimensional crystals of natively secreted LLO were used for structure determination by molecular replacement 30 with Perfringolysin O (PFO) 31 as a search model. Overall, the structure of LLO resembles that of the related CDCs PFO 31 , Anthrolysin O 32 , Intermedilysin 33 , Suilysin 34 and the recently published SLO 35 . LLO is an elongated, rod-like molecule with four distinct domains, referred to as D1 to D4 (Fig.…”

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

“…Domain 3 consists of β-sheets and α-helices. Domain 4 folds into a separate and compact β-sandwich domain (Rossjohn et al, 1997). A cysteine residue is found in a conserved 11-amino-acid sequence (ECT-GLAWEWWR) near the C-terminus in domain 4.…”

“…Molecular modelling has shown that cholesterol binding to this region induces a displacement of the tryptophan rich loop. It is proposed that the high affinity of PFO and also of the cholesterolbinding cytotoxins (K d = 10 nM) for the cholesterol receptor is involved in concentrating the toxin in cholesterol molecules organized into arcs on the target membrane, promoting oligomerization and membrane insertion (Rossjohn et al, 1997). Cholesterol is clustered in lipid membrane microdomains or rafts, and PFO is a useful tool to identify the membrane rafts (Waheed et al, 2001).…”

“…Monomers do not insert their transmembrane hairpins individually, but a cooperation between PFO monomers is required to drive the insertion of the pre-pore complex, which appears to be an all-or-nothing process (Hotze et al, 2002). The charged face of domain 4 amphipathic β-hairpin forms the inner lining of the pore, and the other face is protected from the hydrophobic part of the lipid bilayer by cholesterol molecules (Rossjohn et al, 1997). The role of cholesterol in the pore formation is still speculative.…”

Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes

“…This family of toxins comprises, to date, 23 members from different Gram-positive genera, including perfringolysin (PFO) whose X-ray structure has been solved (Rossjohn et al, 1997), a cytolysin secreted by the extracellular pathogen Clostridium perfringens. Despite structural and functional similarities between LLO and PFO (44 % identity at the peptide level), Portnoy and co-workers (Jones & Portnoy, 1994) have previously shown that the pfo gene, cloned under the control of the promoter phly, was unable to complement an hly mutation in L. monocytogenes for virulence in the mouse model of infection.…”