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J. Biol. Chem., Vol. 282, Issue 10, 7668-7678, March 9, 2007

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Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain^*

C. T. Ranjith-Kumar, William Miller, Jin Xiong¹, William K. Russell^¶, Roberta Lamb^||, Jonathan Santos, Karen E. Duffy^||, Larissa Cleveland, Mary Park, Kanchan Bhardwaj, Zhaoxiang Wu^¶, David H. Russell^¶2, Robert T. Sarisky^||, M. Lamine Mbow^||, and C. Cheng Kao³

From the Department of Biochemistry and Biophysics, Department of Biology, and ^¶Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843 and ^||Discovery Research, Centocor Research and Development, Inc., Radnor, Pennsylvania 19087

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-B 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.

Received for publication, November 28, 2006 , and in revised form, December 28, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by Welch Foundation Grant A1589.

² Supported by National Institutes of Health Grant RR019587, National Science Foundation Grant CHE-0521216, Department of Energy Grant DE-FG02-04ER15520, and Welch Grant A-1176.

³ To whom correspondence should be addressed. Fax: 979-845-9274; E-mail: ckao{at}tamu.edu.

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