Molecular mechanism by which luteolin inhibits toll-like receptor 4 activation: Multi-spectroscopic analysis and molecular simulation

Excessive inflammatory response is an important cause of various diseases. Toll-like receptor 4 (TLR4), a central innate immune receptor, represents a promising target for treating inflammatory diseases. Therefore, the search for natural inhibitors of TLR4 is essential for the prevention and treatment of inflammatory diseases. Luteolin (Lut), a dietary flavonoid with significant anti-inflammatory effects, is a highly promising functional food ingredient. Through integrated spectroscopic characterization and computational simulations, the molecular interaction dynamics between Lut and TLR4 were elucidated in this work. It was found that Lut exhibited concentration-dependent TLR4 fluorescence quenching through static quenching, forming a stable complex spontaneously, speculating that it may bind to the amino acid residues Arg264, Tyr292, Asp294 and Thr319 in TLR4 mainly through electrostatic and hydrogen-bonding interactions. Lut decreased the content of α-helix of TLR4, increased the content of random coil, and decreased its protein compactness and structural instability, thereby inhibiting TLR4 activation. The binding constant between the mutant protein and Lut was significantly decreased by alanine mutagenesis, confirming that Arg264 and Tyr292 are critical residues for the interaction between Lut and TLR4. These findings revealed the molecular mechanism by which Lut regulated the conformational changes of TLR4 and inhibited its activation, providing an important theoretical foundation for exploiting Lut as a natural TLR4-targeted therapeutic agent.