Ranjith-Kumar C T, Miller William, Xiong Jin, Russell William K, Lamb Roberta, Santos Jonathan, Duffy Karen E, Cleveland Larissa, Park Mary, Bhardwaj Kanchan, Wu Zhaoxiang, Russell David H, Sarisky Robert T, Mbow M Lamine, Kao C Cheng
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, USA.
J Biol Chem. 2007 Mar 9;282(10):7668-78. doi: 10.1074/jbc.M610946200. Epub 2007 Jan 5.
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-kappaB 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.
人Toll样受体3（TLR3）胞外结构域（ECD）的结构最近通过X射线晶体学解析得到，由此产生了许多关于TLR3功能的模型（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）。该结构揭示了四对半胱氨酸，推测它们参与二硫键形成，还有几个残基预计参与ECD亚基之间的二聚化，以及可能与聚肌苷酸-聚胞苷酸（poly(I:C)）结合的表面。此外，有两个环从蛋白质的中央螺线管结构中突出。我们检测了重组TLR3 ECD的二硫键形成、poly(I:C)结合和蛋白质-蛋白质相互作用。我们还对全长TLR3中可能影响这些特征的残基进行了80多个突变，并检测了它们对TLR3介导的核因子κB（NF-κB）激活的影响。许多影响TLR3活性的突变也影响了作为野生型TLR3显性负性抑制剂的能力。假定的RNA结合丧失不一定影响显性负性活性。所有结果都支持一个模型，即TLR3的二聚体是结合RNA并激活信号转导的形式。
