title: Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.
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.
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Irvine
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Monique
authors.initials: M
authors.lastname: Gangloff
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Walsh
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David R
authors.initials: DR
authors.lastname: Spring
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Gay
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Bryant
mesh_terms: D000818:Animals; D002460:Cell Line; D057809:HEK293 Cells; D006736:Horses; D006801:Humans; D008024:Ligands; D008070:Lipopolysaccharides / Q000235:genetics / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D013045:Species Specificity; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
chemical_list: C495377:LY96 protein, human; D008024:Ligands; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4