In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd8

Title:In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd8

Volume: 23 Issue: 1

Author(s): Tae-Ho Jang, Kannan Badri Narayanan and Hyun Ho Park

Affiliation:

Keywords: Toll-like receptors, MyD88, TIR domain, Nuclear factor B, Escherichia coli, in vitro.

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 TLRs^TIR with cytosolic adaptor protein, MyD88TIR recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor B (NF-B) and activation protein 1 (AP-1) and other effector molecules. In the present investigation, TLR5^TIR, TLR6^TIR and MyD88^TIR genes were subcloned and overexpressed in bacterium Escherichia coli strain BL- 21 (DE3). The purification and biochemical characterization of TLR5^TIR and TLR6^TIR, and MyD88^TIR 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 TLR6^TIR 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 MyD88^TIR to TLR5/6^TIR, time-dependent aggregation was not observed in both TLRs^TIR at both room temperature and 4 ºC for 2 d, influencing the solubility of TLR5/6^TIR. Moreover, TLR5^TIR 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.

Cite this article as:

Jang Tae-Ho, Narayanan Badri Kannan and Park Ho Hyun, In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd8, Protein & Peptide Letters 2016; 23 (1) . https://dx.doi.org/10.2174/0929866523666151106123613