title: Critical residues involved in Toll-like receptor 4 activation by cationic lipid nanocarriers are not located at the lipopolysaccharide-binding interface.
abstract: DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK. clonez@ulb.ac.be.|Structure and Function of Biological Membranes, Université Libre de Bruxelles, Brussels, Belgium. clonez@ulb.ac.be.
authors.forename: Caroline
authors.initials: C
authors.lastname: Lonez
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Irvine
authors.affiliation: Structure and Function of Biological Membranes, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Malvina
authors.initials: M
authors.lastname: Pizzuto
authors.affiliation: Structure and Function of Biological Membranes, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Boris I
authors.initials: BI
authors.lastname: Schmidt
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Gay
authors.affiliation: Structure and Function of Biological Membranes, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Ruysschaert
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Monique
authors.initials: M
authors.lastname: Gangloff
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Bryant
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006736:Horses; D006801:Humans; D050356:Lipid Metabolism; D018950:Lipopolysaccharide Receptors / Q000502:physiology; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013045:Species Specificity; D051197:Toll-Like Receptor 4 / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D011993:Recombinant Fusion Proteins; D051197:Toll-Like Receptor 4
keywords: Activation mechanism; Cationic lipid; Nanoparticle; Species specificity; Toll-like receptor