Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain

C. T. Ranjith-Kumar, William Miller, Jin Xiong, William K. Russell, Roberta Lamb, Jonathan Santos, Karen E. Duffy, Larissa Cleveland, Mary Park, Kanchan Bhardwaj, Zhaoxiang Wu, David H. Russell, Robert T. Sarisky, M. Lamine Mbow, C. Cheng Kao

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48 Scopus citations

Abstract

The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585; Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980) The structure revealed four pairs of cysteines that are putatively involved in disulfide bond formation, several residues that are predicted to be involved in dimerization between ECD subunits, and surfaces that could bind to poly(I:C). In addition, there are two loops that protrude from the central solenoid structure of the protein. We examined the recombinant TLR3 ECD for disulfide bond formation, poly(I:C) binding, and protein-protein interaction. We also made over 80 mutations in the residues that could affect these features in the full-length TLR3 and examined their effects in TLR3-mediated NF-κB activation. A number of mutations that affected TLR3 activity also affected the ability to act as dominant negative inhibitors of wild type TLR3. Loss of putative RNA binding did not necessarily affect dominant negative activity. All of the results support a model where a dimer of TLR3 is the form that binds RNA and activates signal transduction.

Original languageEnglish (US)
Pages (from-to)7668-7678
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number10
DOIs
StatePublished - Mar 2 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Ranjith-Kumar, C. T., Miller, W., Xiong, J., Russell, W. K., Lamb, R., Santos, J., Duffy, K. E., Cleveland, L., Park, M., Bhardwaj, K., Wu, Z., Russell, D. H., Sarisky, R. T., Mbow, M. L., & Kao, C. C. (2007). Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain. Journal of Biological Chemistry, 282(10), 7668-7678. https://doi.org/10.1074/jbc.M610946200