The Toll-like receptor 4 region Glu24-Pro34 is critical for interaction with MD-2

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS) but requires MD-2, a molecule associated with the extracellular TLR4 domain, to respond efficiently to LPS. The purpose of this study was to determine the critical stretch of primary sequence in the TLR4 region involved in MD-2 recognition. TLR4 and TLR4/2a chimera consisting of the TLR4 region Met1-Phe54 and the TLR2 region Ala53-Ser784 were coprecipitated with MD-2, but the deletion mutant TLR4ΔE24-P34 in which the TLR4 region Glu24-Pro34 was deleted failed to coprecipitate. In agreement with the MD-2 binding, LPS-conjugated beads sedimented TLR4 and TLR4/2a chimera but not TLR2 with MD-2. TLR4ΔE24-P34 barely coprecipitated with LPS-beads. The cells that had been cotransfected with TLR4ΔE24-P34 and MD-2 did not induce NF-κB activation in response to LPS. These results clearly demonstrate that the amino-terminal TLR4 region of Glu24-Pro34 is critical for MD-2 binding and LPS signaling.