Kinetics of Binding of LPS to Recombinant CD14, TLR4, and MD-2 Proteins

TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants ($K_D$) of LPS for immobilized CD14 and MD-2 were $8.7{\mu}m$, and $2.3{\mu}m$, respectively. The association rate constant ($K_{on}$) of LPS for MD-2 was $5.61{\times}10^3M^{-1}S^{-1}$, and the dissociation rate constant ($K_{off}$) was $1.28{\times}10^2S^{-1}$, revealing slow association and fast dissociation with an affinity constant $K_D$ of $2.33{\times}10^6M$ at $25^{\circ}C$. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex.