期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 281, 期 40, 页码 30132-30142出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M602057200
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资金
- NIAID NIH HHS [R01 AI034358, R01 AI034358-10, R01-AI34358] Funding Source: Medline
- NIDDK NIH HHS [DK 56703] Funding Source: Medline
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 alpha 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 PAM(3)CSK(4) mediated NF-kappa 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.
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