In vitro reconstitution of the Toll/interleukin-1 receptor (TIR) domain complex between TLR5/6 and MyD88.

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, MyD88(TIR) recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor kappa B (NF-kappa 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, MyD88(TIR) interacted with TLR5(TIR), 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 degrees C 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.