TLR4 is involved in lipopolysaccharide (LPS) recognition, a major cell wall component of Gram-negative bacteria. MD-2 is a molecule that is essential for the cellular distribution of TLR4 and it associates with the extracellular domain of TLR4. The TLR4/MD-2 complex binds LPS. In addition to LPS, TLR4 can recognize several other ligands such as: taxol, envelope proteins, fusion proteins; and endogenous ligands, such as: HSP60, HSP70, oligosaccharides of hyaluronic acid and fibrinogen.? Our goal is to determine the high resolution crystal structure of human TLR4 (hTLR4) and hTLR4 in complex with hMD-2. Initial attempts to express hTLR4 and hMD-2 in E. coli were unsuccessful due to the lack of glycosylation that is required for correct folding and secretion of these proteins. Therefore, the baculovirus expression system was used, that provides correct glycosylation, folding and secretion of the target proteins. Expression of hTLR4 and hMD-2 under the control of the polyhedron promoter with the N-terminal GP67 secretion signal and C-terminal affinity tags (FLAG-tag and His-tag) was tested in both SF9 and Hi5 cells. Hi5 cells produce more target protein and were used in large scale expressions. The protein secreted to the media was purified using two steps of affinity chromatography, followed by a size exclusion step. The concentrated, pure protein was used in extensive crystallization trials with a Mosquito crystallization robot. However, up to now we were unable to obtain any crystals of the hTLR4, hMD-2, or hTLR4/hMD-2 complex. Constructs for mouse TLR4 (mTLR4) and mMD-2 were generated in a similar way to those of hTLR4 and hMD-2. The expressed and purified mTLR4 and mMD-2 were subjected to extensive crystallization trials without success. Co-expression for both human and mouse TLR4/MD-2 was performed, the complex purified and used in crystallization trials. The stable TLR4/MD-2 complex was further complexed with ultrapure LPS or monophosphoryl lipid A, and used in crystallization trials. None of these constructs have yielded crystals and we are pursuing further crystallization trials..
