Proteins Of Innate Immunity: Tlr3 and dsRNA binding.

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have investigated the molecular mechanism by which Toll-Like Receptor 3 (TLR3) recognizes double stranded RNA (dsRNA). In 2005, we succeeded in determining the molecular structure of human TLR3 ectodomain. Since then, numerous reports have given different models of TLR3 recognizing dsRNA based on biochemistry and cell molecular result. In all the proposed models TLR3 is considered to form a dimer to bind dsRNA. However, until now no precise molecular model has been demonstrated. We have been using mouse TLR3 instead of human TLR3. Our specific aims are a) to produce and crystallize mouse TLR3 ectodomain and to determine its structure by X-ray analysis, comparing mouse with human TLR3 ectodomain structures; b) to characterize the interaction of mouse TLR3-ECD protein with dsRNA in the crystal, explaining the mechanism of TLR3 recognizing antigen in vertebrate immune system.[unreadable] [unreadable] mTLR3-ECD fused to an N-terminal GP67 secretion signal sequence and a C-terminal TEV cleavage site followed by 6xHis and strep tags was inserted into a baculovirus expression system by the Protein Expression Laboratory (Science Applications International Corp., Frederick, MD) and expressed in High Five cells (Invitrogen). After optimization of temperature and insect cell culture medium, we finally achieved a high expression of 6mg/L for mTLR3 L. Crystals were obtained that diffracted to 2.7A and the crystal stucture revealed a close homology with the human TLR3. [unreadable] [unreadable] Using different size dsRNA molecules, both the in vivo and in vitro experiments gave the same minimum size dsRNA which can bind and activate TLR3 for the downstream signaling, approximately 45 base pairs. We made a complex of mTLR3 with the minimum size dsRNA, and have been able to produce crystals of size 0.04x0.04x0.02mm, which can be diffracted to 3.4A. The crystal structure showed that the dsRNA was in the form of a straight helix making contact with two TLR3 ectodomains at two separate regions on each ectodomain which were confirmed by mutation. TLR3 contacts the sugar phosphate backbones of the RNA and makes no contact with the bases, thus accounting for the absence of sequence specificity. Additional interactions between the two C-terminal capping regions formed the dimer and the contact brought the two C-termini of the ectodomain close so that the cytoplasmic TIR domains could dimerize, believed to be the initiation event in the signaling process.[unreadable] [unreadable] Dimerization of the cytoplasmic TIR domains creates the signaling platform for adaptor recruitment. The adaptor protein used by TLR3 signaling is TRIF (TIR-domain-containing adaptor protein inducing IFN). It is hypothesized that TRIF recruitment depends on homotypic TIR domain interactions. To address the initiation mechanism for cytoplasmic TLR3 signaling, we are: 1) Producing and attempting to crystallize the TLR3-TIR domain (TIR3), determine its structure by X-ray analysis, and localize TIR3 dimerization interface. 2) Expressing TRIF, and attempting to crystallize and determine the structure of TIR3-TRIF complex, thus characteriz the interactions between them.