Molecular mechanisms of LPS transfer to TLR4-MD-2 via LBP and CD14

Abstract

Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex plays a critical role in host pro-tection against Gram-negative bacterial infection. Recognition of lipopolysaccharide (LPS), a toxic compo-nent of the outer membrane of Gram-negative bacteria, by TLR4-MD-2 evokes innate immune response. Lipopolysaccharide-binding protein (LBP) and cluster of differentiation 14 (CD14) sequentially mediate LPS transport from LPS aggregate to TLR4-MD-2. Although crystal structure of LBP, CD14, and TLR4-MD-2 complex have been determined, molecular mechanisms underlying the LPS transfer cascade remain unclear. To elucidate the mechanisms, we characterized dynamic interactions among the proteins and LPS in each intermediate step of the LPS transport process using negative-stain transmission electron microscopy (TEM) and single molecule fluorescence analysis. We found that LBP bound longitudinally to the surface face of LPS micelle via its six basic residues (Lys-67, Lys-68, Arg-73, Arg-119, Lys-120, and Lys-124) in its N-terminal tip. The binding of LBP to LPS micelle was required for interaction of CD14 with LBP. Notewor-thy, while a LBP stayed in binding to a LPS micelle, two basic residues (Lys-319 and Arg-322) in the C-terminus of LBP interacted with two acidic amino acids (Asp-251 and Asp-297) on the concave surface of CD14. A LPS micelle-bound LBP transferred a LPS molecule to CD14 monomer repetitively without detach-ing from the micelle. Repulsion between Asp-311 of LBP and an acidic patch in concave region of N-terminus of CD14 resulted in swift detachment of CD14 from LBP after obtaining LPS. In addition, we found that LRR13 and LRR14 of TLR4 were essential for transport of LPS from CD14 to TLR4-MD-2 complex. Collectively, our results provide structural and molecular mechanistic insights into the LPS transport from LPS micelles to TLR4-MD-2 via LBP and CD14.