Proteins Of Innate Immunity: Tlr3 and dsRNA binding.

Diabetes, Digestive, Kidney Diseases

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Abstract

We are investigating the molecular mechanism by which Toll-Like Receptor 3 (TLR3) recognizes double stranded RNA (dsRNA). In 2005, our lab 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 have been obtained that diffract to 3.5A. [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 active TLR3 for the downstream signaling. 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 5.0 .[unreadable] [unreadable] To reach our goal, we will still work on the crystallization of both the mTLR3 itself and complex of mTLR3 and dsRNA. We will compare the structure of mTLR3 and hTLR3 to see whether the conservation in amino sequence consists with the structure. And we need the three dimension structure to give the molecular model of how TLR3 recognizing its natural ligand dsRNA.