Molecular Dynamics Study of Lipid and Cholesterol Reorganization Due to Membrane Binding and Pore Formation by Listeriolysin O.

Abstract

Pore-forming toxins are proteinsexpressed by bacteria to primarily cause infections in the host cell. Cholesterol-dependent cytolysins (CDCs) are a class of proteins whose pore-forming ability requires the presence of cholesterol in the membrane. Upon binding to the target cell, cholesterol-recognizing residues in the membrane binding D4 subdomain assist in stabilizing both the pre-pore and pore states which occur during protein oligomerization on the cell membrane. Super resolution-stimulated emission depletion (STED) microscopy experiments (Sarangi et al. in Langmuir, 32:9649-9657, 2016) on supported lipid bilayers have shown that listeriolysin (LLO), a CDC expressed by Listeria monocytogenes, a food-borne pathogen, induces both spatial and dynamic heterogeneity in bilayer membranes. Here, we use all-atom molecular dynamics simulations to explore molecular details of the induced membrane reorganization by considering two distinct states of the oligomerized LLO protein in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol membrane. In the membrane bound (MB) state, four D4 subunits are placed at the bilayer interface in a pre-pore configuration and the membrane-inserted (MI) state consists of a tetrameric arc-like pore configuration. By analyzing lipid-order parameters, mobilities, and diffusion coefficients, we examine the induced spatial heterogeneity that occurs in both the MB and MI states. This heterogeneity is primarily driven by the local density enhancement of cholesterol in the vicinity of the MB, D4 subunits leading to distinct differences in lipid and cholesterol mobility across the two leaflets as well as enhanced lipid mobilities in regions where cholesterol is depleted. The leaflet-induced heterogeneity is greater for the MB state when compared with the MI state and the dynamic variations are more pronounced in the extracellular leaflet when compared with the cytosolic leaflet. Our study provides molecular-level insights into the inhomogeneity and perturbation induced in bilayer membranes upon LLO binding and pore formation and is expected to represent trends across PFTs in the broad CDC subclass of proteins.