Abstract
Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.
Highlights
Toll-like receptor 4 (TLR4) represents a major target for vaccine adjuvants, and inhibition may help to treat TLR4 over-stimulation in bacterial sepsis[10]
We show that Δ Gc is more favourable than Δ Go in the case of LPA, but that this trend is reversed for lipid IVa (LPIVa) and Erit; in other words, agonist is biased towards binding to the conformation of MD-2 associated with TLR4 activation, whereas the antagonist preferentially binds to the inactive state
We first used HEK293 cells transfected with components of the human TLR4 pathway and a reporter assay to test for pharmacological effects of LPS, LPIVa, or both in combination (Fig. 2)
Summary
TLR4 represents a major target for vaccine adjuvants, and inhibition may help to treat TLR4 over-stimulation in bacterial sepsis[10]. Its biosynthetic precursor lipid IVa (LPIVa) – which only differs by containing four instead of six acyl chains in its lipid A component (Fig. 1A) – is an inhibitor of human TLR46 and has been a candidate for clinical development in sepsis treatment. Clamshell motions may be allosterically transmitted to a phenylalanine residue (Phe126) at the tip of this loop, so that binding of inhibitors such as LPIVa result in transition from the MD-2c to MD-2o conformation, destabilizing the interaction between MD-2 and TLR4 in the active receptor complex. LPS agonists will tend to be bound to the MD-2c conformation, and stabilize the active receptor complex Antagonists such as LPIVa will favour MD-2o, inhibiting receptor complex formation and signal transduction, whilst competitively blocking its binding site. Being able to predict these equilibria would facilitate rational design of therapeutics targeted towards TLR4
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