title: Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition.
abstract: The receptor complex resulting from association of MD-2 and the ectodomain of Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signal transduction across the cell membrane. We prepared a tertiary structure model of MD-2, based on the known structures of homologous lipid-binding proteins. Analysis of circular dichroic spectra of purified bacterially expressed MD-2 indicates high content of beta-type secondary structure, in agreement with the structural model. Bacterially expressed MD-2 was able to confer LPS responsiveness to cells expressing TLR4 despite lacking glycosylation. We identified several clusters of basic residues on the surface of MD-2. Mutation of each of two clusters encompassing the residues Lys(89)-Arg(90)-Lys(91) and Lys(125)-Lys(125) significantly decreased the signal transduction of the respective MD-2 mutants either upon co-expression with TLR4 or upon addition as soluble protein into the supernatant of cells overexpressing TLR4. These basic clusters lie at the edge of the beta-sheet sandwich, which in cholesterol-binding protein connected to Niemann-Pick disease C2 (NPC2), dust mite allergen Der p2, and ganglioside GM2-activator protein form a hydrophobic pocket. In contrast, mutation of another basic cluster composed of Arg(69)-Lys(72), which according to the model lies further apart from the hydrophobic pocket only weakly decreased MD-2 activity. Furthermore, addition of the peptide, comprising the surface loop between Cys(95) and Cys(105), predicted by model, particularly in oxidized form, decreased LPS-induced production of tumor necrosis factor alpha and interleukin-8 upon application to monocytic cells and fibroblasts, respectively, supporting its involvement in LPS signaling. Our structural model of MD-2 is corroborated by biochemical analysis and contributes to the unraveling of molecular interactions in LPS recognition.
authors.affiliation: Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Gruber
authors.forename: Mateja
authors.initials: M
authors.lastname: Mancek
authors.forename: Hermann
authors.initials: H
authors.lastname: Wagner
authors.forename: Carsten J
authors.initials: CJ
authors.lastname: Kirschning
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010196:Pancreatic Elastase; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D033921:Vesicular Transport Proteins
chemical_list: D000596:Amino Acids; D000954:Antigens, Surface; D002352:Carrier Proteins; D006023:Glycoproteins; D016209:Interleukin-8; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; C104585:NPC2 protein, human; D010455:Peptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D033921:Vesicular Transport Proteins; D010196:Pancreatic Elastase; C017130:cholesterol-binding protein