Gram-negative bacterial endotoxin (i.e lipopolysaccharide; LPS) is one of the most potent stimulants of the innate immune system, which is recognized by the TLR4/MD-2 complex. Binding of LPS triggers formation of the active homodimer TLR4/MD-2, which initiates intracellular signal transmission via the intracellular TIR domain of TLR4. RP105 is a TLR homolog lacking an intracellular TIR domain. In parallel with TLR4, whose signaling depends on the secreted extracellular protein MD-2, the full function of RP105 is dependent on co-expression of the MD-2 homolog, MD-1. MD-1, in contrast to MD-2, does not bind LPS. RP105 is a specific inhibitor of TLR4 signaling in HEK 293 cells, a function conferred by its extracellular domain. RP105 and its helper molecule, MD-1, interact directly with the TLR4 signaling complex, inhibiting its ability to fully respond to the microbial ligand. Moreover, RP105-regulated TLR4 signaling in dendritic cells, as well as endotoxin responses in vivo, label RP105 as a physiological negative regulator of TLR4 responses. The molecular model of the active TLR4/MD-2/LPS homodimer, which was suggested by our research team and afterwards confirmed by the determination of its crystal structure, provides us with an excellent base to investigate the molecular mechanism of negative regulation of TLR4 signaling by RP105 and its co-receptor MD-1. Briefly, the lipid A moiety of monomeric LPS binds to the hydrophobic pocket of MD-2, which causes dimerization of the TLR4 membrane receptor mediated by two binding sites on the extracellular domain of TLR4. Based on the homology between the RP105/MD-1 and TLR4/MD-2 pairs and known biochemical data, we proposed a hypothesis that the RP105/MD-1:TLR4/MD-2 heterodimerizes and prevents activation due to the lack of TIR domain in RP105. To test our hypothesis, we prepared a chimeric protein where the TIR domain of TLR4 is attached to the ectodomain of RP105. We found that the chimeric protein acts as an even better inhibitor of TLR4 signaling than RP105 alone, which tells us that lack of the TIR domain in RP105 is not the only factor for its inhibitory effect. On the basis of our experimental results, we proposed a revised model of TLR4/MD-2:RP105/MD-1 interaction.
COBISS.SI-ID: 4829210