Structural and Thermodynamic Insights into Bacterial Outer Membrane Lipid Signaling by the Innate Immune System

Abstract

Lipopolysaccharide (LPS) from bacterial outer membranes is a potent early indicator of microbial infection and the primary inducer of fatal septic shock syndrome. Its recognition by the immune system is carried out by Toll-like receptor 4 (TLR4) when associated with its co-receptor MD-2, an immunoglobulin-like protein. MD-2 adopts a characteristic “beta-cup” fold with a large hydrophobic cavity, and is able to bind a variety of lipid species. Subtle alterations in the structure of LPS derivatives can profoundly alter the resultant immunological response, hampering the rational design of TLR4 immunomodulators. To unravel the associated structure-activity relationships, we have performed long-timescale, all-atom molecular dynamics simulations and free-energy calculations of the isolated MD-2 co-receptor and the entire signaling-active receptor complex in the presence of a variety of LPS species, as well as an LPS membrane. Unbiased simulations revealed that the MD-2 cavity is highly conformationally flexible, identifying spontaneous switching between active signalling-competent and inactivated states dependent upon the presence of different ligands, leading us to propose a conserved receptor activation mechanism. To gain insights into the thermodynamic determinants of endotoxin recognition, extensive umbrella sampling has been applied to estimate the potential of mean force (PMF) for the binding of LPS molecules to MD-2 co-receptor. Strikingly, stronger binding to signalling-inactivate MD-2 were observed for antagonists, and conversely, stronger binding to active MD-2 for agonists. Comparison of this data to the first ever PMF calculated for extraction of LPS from a model of the bacterial outer membrane has revealed how MD-2 creates a “membrane-like” environment within its protein cavity, providing a mechanism for sensitive LPS recognition by the innate immune system.