The innate immune system defends the host primarily against invading microbes. Toll-like receptors (TLRs), key molecules in the innate immunity, stimulate innate immune responses upon recognition of pathogen-associated molecular patterns (PAMPs). Among 13 TLRs, TLR4 recognizes lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria in the presence of MD-2, then activates the production of inflammatory cytokines. Two accessory proteins, LBP and CD14, facilitate the LPS recognition of TLR4 and rapid induction of inflammatory response. However, severe bacterial infection can exaggerate immune responses, resulting in multiple organ failure and death. Thus, both clearance of the bacteria by sensitive sensing of LPS in the presence of small amount of bacteria and attenuation of the recognition sensitivity in the existence of large number of bacteria are critical for host survival. In order to modulate the sensitivity of LPS recognition, mechanisms of LPS transfer from LPS aggregates to TLR4-MD-2 complex through LBP and CD14 should be elucidated.
To unveil mechanisms underlying the LPS delivery, we characterized dynamic intermediate complexes using negative-stain transmission electron microscopy (TEM) and total internal reflection fluorescence microscopy (TIRFM). We visualized longitudinal binding of LBP to LPS micelle and identified critical residues of LBP for the binding. The structure of LBP-CD14 complex and mechanisms and kinetics of their association and dissociation were also revealed. Interaction interfaces in the complexes were verified using mutants that designed based on crystal structure of the proteins and our negative-stain TEM results. Lastly, mechanism of LBP-mediated LPS transfer to CD14 and TLR4-dependent LPS transfer from CD14 to TLR4-MD-2 were defined. Our findings provide the structural and mechanistic insights into LPS transfer from LPS micelles to TLR4-MD-2.