A new model for pore formation by cholesterol-dependent cytolysins.

Cyril F Reboul,James C Whisstock,Michelle A Dunstone,Nir Ben-Tal

doi:10.1371/journal.pcbi.1003791

Abstract

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.

Highlights

Cholesterol dependent cytolysins (CDCs) represent a major branch of the CDC/membrane attack complex/perforin-like (MACPF) protein superfamily
Identified as virulence factors produced by Gram positive pathogens, CDC toxins have recently been identified in Gram negative bacteria such as Desulfobulbus propionicus and Enterobacter lignolyticus [1,2]
By first aligning the whole molecules we identified a major rigid body consisting of Domains 1, 3 and the upper part of Domain 2 close to Domain 1 and packing against TMH1 (Figure 2A,B)

Summary

Introduction

Cholesterol dependent cytolysins (CDCs) represent a major branch of the CDC/membrane attack complex/perforin-like (MACPF) protein superfamily. Pore formation is associated with a variety of toxic functions, including escape from the intracellular phagolysosome (LLO) [3] and the delivery of folded toxins such as nicotinamide adeninedinucleotide-glycohydrolase by SLO [4]. The first crystal structure of a monomeric CDC (PFO) suggested that the molecule comprises four distinct domains. The mechanism of CDC membrane insertion has been well characterized and mapped to the structure. During pore formation two clusters of helices (Transmembrane Helix 1 (TMH1) and 2 (TMH2)) within Domain 3 unwind and insert into the membrane as two amphipathic b-hairpins. Domain 2, a region unique to CDCs, essentially comprises an elongated three-stranded b-sheet that links the pore forming head domain (Domains 1 and 3) to Domain 4. Domain 4 contains the determinants for interacting with the membrane, including a key conserved sequence that is important for binding cholesterol [7]

Methods

Results

Conclusion