The FASEB Journal • Research Communication Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2

ABSTRACT Toll-like receptors (TLRs) belong to the family of pattern recognition receptors, as they recog-nize molecules sharing a broad structural pattern rather than a single defined structure. Bacterial LPS is recog-nized by MD-2, which is associated with the extracellu-lar 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 recog-nition 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 nongly-cosylated 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 hydropho-bic patch and a pair of separated negative charges.— Manček-Keber, M., Jerala, R. Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligan