Abstract
The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.
Highlights
The Toll-like receptor (TLR) family is the most widely studied pattern recognition receptor family, comprising 13 mammalian members, of which 10 are expressed in humans [1]
A key question generated by solving the LPS-bound Toll-like receptor 4 (TLR4)/ MD-2 structures was whether there would be similar molecular determinants when other lipid A variants bound to the receptor complex [4]
Human and horse TLR4 and MD-2 are more closely related to one another than either are to the mouse receptors, yet Rhodobacter sphaeroides LPS (RSLPS) is an agonist at horse TLR4/MD-2 and an antagonist at both the mouse and human receptors
Summary
The Toll-like receptor (TLR) family is the most widely studied pattern recognition receptor family, comprising 13 mammalian members, of which 10 are expressed in humans [1]. Toll-like receptor 4 (TLR4) is expressed on the plasma membrane and endosomes to sense lipopolysaccharide (LPS) from Gram-negative bacteria, requiring the co-receptor MD-2 to bind the lipid component of LPS (lipid A) and form the active complex [2,3]. TLR4/MD-2 from all domestic mammalian species recognises hexaacylated lipid A from E. coli (figure 1A) as an agonist, but structural variability in lipid A from other Gram-negative organisms alters their efficacy (maximum stimulation, Emax) and potency (half maximum effective concentration, EC50) at the receptor complex. A reduction in acyl chain number from six to four in the lipid A synthesis intermediate lipid IVa (figure 1B) makes this compound an antagonist at TLR4/MD-2, but only in humans. A tetraacyl chain lipid A analogue derived from the structure of Rhodobacter sphaeroides LPS (RSLPS), is an antagonist at human, mouse and horse TLR4/MD-2
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