Key residues in TLR4-MD2 tetramer formation identified by free energy simulations.

Toll-like receptors (TLRs) play a central role in both the innate and adaptive immune systems by recognizing pathogen-associated molecular patterns and inducing the release of the effector molecules of the immune system. The dysregulation of the TLR system may cause various autoimmune diseases and septic shock. A series of molecular dynamics simulations and free energy calculations were performed to investigate the ligand-free, lipopolysaccharide (LPS)-bound, and neoseptin3-bound (TLR4-MD2)(2) tetramers. Compared to earlier simulations done by others, our simulations showed that TLR4 structure was well maintained with stable interfaces. Free energy decomposition by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method suggests critical roles that two hydrophobic clusters I-85-L-87-P-88 and I-124-L-125-P-127 of MD2, together with LPS and neoseptin3, may play in TLR4 activation. We propose that 1) direct contacts between TLR4 convex surface and LPS and neoseptin3 at the region around L-442 significantly increase the binding and 2) binding of LPS and neoseptin3 in the central hydrophobic cavity of MD2 triggers burial of F-126 and exposure of I-85-L-87-P-88 that facilitate formation of (TLR4-MD2)(2) tetramer and activation of TLR4 system. Author summary Toll-like receptors (TLRs) play a central role in both the innate and adaptive immune systems and its dysregulation may cause a host of serious and often life-threatening diseases. A great deal has been known about this system. Yet, how exactly this system works and which part is responsible to activate the system remains elusive. This work seeks to identify the key parts of the system that play roles in initiating the signaling cascade. The knowledge gained from this study is expected to shed light to how this important system works which in turn may help design more effective and life-saving anti-inflammatory and anti-cancer drugs.