Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2.
Toll-like receptors (TLRs) belong to the family of pattern recognition receptors, as they recognize molecules sharing a broad structural pattern rather than a single defined structure. Bacterial LPS is recognized by MD-2, which is associated with the extracellular domain of TLR4. Understanding the molecular recognition pattern of MD-2 could lead to efficient inhibitors of the excessive LPS signaling needed for early treatment of sepsis. The effect of the acyl chain variability of lipid A on its biological activity indicates that in addition to electrostatic interactions, the recognition must also involve hydrophobic interactions. We show that the fluorescent hydrophobic probe bis-ANS binds to MD-2 with a dissociation constant in the 10 nanomolar range, both to glycosylated and to nonglycosylated MD-2, and requires its native conformation. The binding site of bis-ANS overlaps with the binding site of LPS and is in the proximity of the single tryptophan residue. Furthermore, photoincorporation of bis-ANS by UV light inhibits the ability of MD-2 to confer the LPS responsiveness to the TLR4-transfected HEK293 cell line. Our results show that the structural pattern recognized by MD-2 is defined by the hydrophobic patch and a pair of separated negative charges.
Mancek-Keber
Mateja
M
Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Jerala
Roman
R
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007668:Kidney; D008024:Ligands; D008050:Lipid A / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D051193:Toll-Like Receptors / Q000502:physiology; D014162:Transfection
D005456:Fluorescent Dyes; C495377:LY96 protein, human; D008024:Ligands; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D011994:Recombinant Proteins; D051193:Toll-Like Receptors