The Toll-like receptors (TLRs) comprise a family of at least 10 integral membrane receptors that are essential for initiating inflammatory reactions in response to pathogens such as viruses and bacteria. The major objective of this research is to elucidate the structures of external domains of TLRs in order to learn how they interact with pathogen associated molecular patterns (PAMP). TLR4 is required for responsiveness to lipopolysaccharide (LPS) and lipoteichoic acid (LTA); TLR4 deficient mice are resistant to septic shock but are highly susceptible to infection by Gram negative bacteria. TLR4 forms a complex with MD-2, a secreted protein that binds to the extracellular domain of TLR4, and MD-2 is required for a functional TLR4. We initially attempted to produce TLR4 extracellular domain in bacteria, the simplest expression system, but were unsuccessful in refolding the protein from inclusion bodies. Subsequently we have established a secretion system using S2 insect cells, because these cells can correctly fold proteins in their ER, can secrete large amounts of native protein and are easy to grow. We have now created a stable S2 line that secretes MD-2, establishing that the system works in our hands. The TLRs contain typical transmembrane regions connected to characteristic cytoplasmic signaling domains of about 200 residues that also share homology with the cytoplasmic domains of the IL-1 and IL-18 receptors and are known as TIR (Toll-IL-1R) domains. MyD88, the first downstream components in the Toll-IL-1R signaling pathway, also contains a TIR domain. The X-ray structures of the TIR domains of TLRs 1 and 2 have been previously determined, and consist of globular protein with a well defined face that can interact with the TIR domains of MyD88. Currently, we have small crystals of the TIR domain of MyD88, produced in bacteria, and we intend to determine its three dimensional structure, preferably complexed with the TIR domains of TLRs. This, along with solutions studies would show directly how TLRs interact with the first component of the TLR/IL-1R core signaling pathway.