title: In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd88.
abstract: Toll-like receptors (TLRs) are evolutionarily conserved receptors with trimodular structure to respond to endogenous ligands and exogenous ligands from microbial pathogens. The highly conserved cytoplasmic C-terminal Toll/interleukin-1 receptor (TIR) domain of TLRs plays a crucial role in inflammatory reactions. In myeloid differentiation primary-response protein 88 (MyD88)- dependent signaling pathway, the interaction of TLRsTIR with cytosolic adaptor protein, MyD88TIR recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor kB (NF-kB) and activation protein 1 (AP-1) and other effector molecules. In the present investigation, TLR5TIR, TLR6TIR and MyD88TIR genes were subcloned and overexpressed in bacterium Escherichia coli strain BL- 21 (DE3). The purification and biochemical characterization of TLR5TIR and TLR6TIR&, and MyD88TIR proteins were also performed. The protein-protein interactions between TIR domains of TLR5 and TLR6 with MyD88, respectively, were evaluated in vitro at physiological pH and salt concentration. The in vitro reconstitution results showed that under physiological pH and salt concentration, MyD88TIR interacted with TLR5TIR, and did not interact with TLR6TIR protein. Both TIR domain-containing TLR5 and TLR6 proteins were prone to aggregation in a temperature-dependent manner at room temperature. At normal physiological pH and salt concentration, with the addition of binding partner MyD88TIR to TLR5/6TIR, time-dependent aggregation was not observed in both TLRsTIR at both room temperature and 4 ºC for 2 d, influencing the solubility of TLR5/6TIR. Moreover, TLR5TIR alone exhibited increase in solubility of the protein with increase in the salt concentration of the buffered solution from 0.025 M to 1.25 M at room temperature.
authors.forename: Tae-Ho
authors.initials: TH
authors.lastname: Jang
authors.forename: Kannan Badri
authors.initials: KB
authors.lastname: Narayanan
authors.affiliation: School of Biotechnology and Graduate School of Biochemistry, Yeungnam University, Gyeongsan 712-749, South Korea. hyunho@ynu.ac.kr.
authors.forename: Hyun Ho
authors.initials: HH
authors.lastname: Park
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D053594:Myeloid Differentiation Factor 88 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066329:Protein Aggregates; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054712:Salinity; D013696:Temperature; D051198:Toll-Like Receptor 5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051216:Toll-Like Receptor 6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D066329:Protein Aggregates; C495346:TLR5 protein, human; C495368:TLR6 protein, human; D051198:Toll-Like Receptor 5; D051216:Toll-Like Receptor 6