Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation

Abstract

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.

Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

“…time scales up to a few microseconds, have to a limited extent been used to illustrate changes in lipid dynamics and conformational transitions in the presence of pore-forming proteins (26)(27)(28) as well as antimicrobial peptides (29,30). However, connecting the structure of the membrane-bound oligomeric intermediates to leakage and ensuing lipid reorganization is challenging, as it requires simultaneous monitoring of several quantities to yield a complete picture of pore-formation dynamics.…”

Membrane perforation by the pore-forming toxin pneumolysin

“…Despite the limitations of the coarse-grained models imposed by the fixed protein geometry (24, 35), the coarse-grained simulations capture the cholesterol dependence of PLY membrane docking, a defining feature of the CDC family. Nevertheless, it is possible that cholesterol has additional stabilizing functions (36), as required for pore formation of alpha-toxins (37) or for dimerization of GPCRs (38).…”

High-resolution cryo-EM structures of the E. coli hemolysin ClyA oligomers

“…We then tested BTLE liposomes with different lipid composition and did find pore-like structures ( Fig 4A ). Cholesterol has been revealed to facilitate membrane binding and pore complex stabilization of ClyA [22, 23]. Cholesterol is not listed as a component on the manufacturer’s data sheet, but approximately 60% of BTLE ingredients are unknown.…”

“…ClyA is capable of inducing leakage of liposomes with a simple composition of POPC and DOPS. Cholesterol has been revealed to facilitate membrane binding and pore complex stabilization [22, 23], which may explain the pore formation of ClyA on BTLE liposomes but not POPC-DOPS liposomes. By separating the liposome leakage activity from the pore-forming activity, we demonstrate that binding of ClyA is sufficient to cause liposome leakage of small molecules without the formation of mature pores on liposome membranes.…”