Snyder Greg A, Deredge Daniel, Waldhuber Anna, Fresquez Theresa, Wilkins David Z, Smith Patrick T, Durr Susi, Cirl Christine, Jiang Jiansheng, Jennings William, Luchetti Timothy, Snyder Nathaniel, Sundberg Eric J, Wintrode Patrick, Miethke Thomas, Xiao T Sam
From the Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 2014 Jan 10;289(2):669-79. doi: 10.1074/jbc.M113.523407. Epub 2013 Nov 25.
The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys(89) and Cys(134). A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.
Toll/白细胞介素-1受体（TIR）结构域是至关重要的天然免疫信号传导模块。含微生物TIR结构域的蛋白通过分子模拟抑制Toll样受体（TLR）信号传导。布鲁氏菌的含TIR结构域蛋白TcpB通过与宿主衔接蛋白TIRAP/Mal和MyD88相互作用来抑制TLR信号传导。为了表征宿主蛋白的微生物模拟，我们测定了布鲁氏菌蛋白TcpB和宿主衔接蛋白TIRAP的TIR结构域的X射线晶体结构。我们进一步利用氢/氘交换质谱对TcpB的同型相互作用进行了表征，并通过免疫共沉淀和NF-κB报告基因检测对TcpB与TIRAP的异型相互作用进行了表征。TcpB TIR结构域的晶体结构揭示了包含BB环的微管结合位点以及由DD环和EE环介导的对称二聚体。通过氢/氘交换质谱进行的肽图分析验证了这种二聚化界面。人TIRAP TIR结构域的晶体结构揭示了一种独特的N端TIR结构域折叠，其中包含由半胱氨酸（89）和半胱氨酸（134）形成的二硫键。TcpB和TIRAP晶体结构之间的比较揭示了包含BB环的区域存在显著的构象差异。这些发现强调了微生物和宿主TIR结构域中分子特征的异同，这提示了细菌模拟宿主信号衔接蛋白（如TIRAP）的机制。
