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J. Biol. Chem., Vol. 281, Issue 40, 30132-30142, October 6, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/40/30132    most recent M602057200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gautam, J. K. Articles by Smith, M. F. Search for Related Content PubMed PubMed Citation Articles by Gautam, J. K. Articles by Smith, M. F., Jr. Social Bookmarking                      What's this?

Structural and Functional Evidence for the Role of the TLR2 DD Loop in TLR1/TLR2 Heterodimerization and Signaling^*

Jitendra K. Gautam, Ashish, Laurey D. Comeau, Joanna K. Krueger, and Michael F. Smith, Jr.^¶1

From the Departments of Medicine, Digestive Health Center of Excellence, and ^¶Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908, and the Department of Chemistry, University of North Carolina at Charlotte, Charlotte, North Carolina 28223

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the D region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM₃CSK₄ mediated NF-B activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.

Received for publication, March 3, 2006 , and in revised form, June 22, 2006.

^* This work was supported by grants from the National Institutes of Health (R01-AI34358), the American Heart Association (to M. F. S.), and the Immunology/Cell Isolation Core of the UVA Silvio O. Conte Digestive Health Research Center (DK 56703). 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.

¹ To whom correspondence should be addressed: University of Virginia Health System, PO Box 800708, Charlottesville, VA 22908-0708. Tel.: 434-924-2573; Fax: 434-243-9645; E-mail: mfs3k{at}virginia.edu.

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