Direct effects of ethanol at relevant concentrations on conformation and ligand binding of Toll-like Receptor 3 (TLR3)

Abstract

Abstract The purpose of this study was to seek direct physical evidence that ethanol at concentrations relevant to binge drinking alters the conformation of TLR3 and decreases ligand binding. We and others have reported that TLR3-mediated responses are significantly suppressed by concentrations of ethanol that occur in binge drinkers (J. Immunol., 2004, 173: 2715–2724). This suppression can be detected at all levels of signaling that have been tested (from MAP kinases to NF-kB), but it remains possible that ethanol acts directly on TLR3 and that its other effects are secondary to altered TLR3 conformation. Purified ectodomains of TLR3 were used in this study, because they comprise most of the TLR3 molecule, and dimerization of TLR3 ectodomains is necessary for signal transduction and activation of TLR3-mediated innate immunity. Using circular dichroism (CD) to measure changes in protein conformation, we demonstrated that ethanol concentrations from 20–100 mM substantially altered the conformation of human and mouse TLR3 ectodomains complexed with a defined dsRNA ligand. Binding of poly I:C, a dsRNA analog, was significantly inhibited by ethanol. Modeling with Autodock 4 software indicated that mouse and human TLR3 contain a putative ethanol binding site with a dissociation constant consistent with the concentration dependence of the conformational changes. These results support the hypothesis that ethanol inhibits TLR3-mediated functions by directly altering the conformation of TLR3. This work was supported by NIH R01AA09505 and SBP was supported by NIH grant P20GM103646.