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: Anton Gruber, Mateja Mancek, Hermann Wagner, Carsten J Kirschning, Roman Jerala
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 27 Pg. 28475-82 (Jul 02 2004) ISSN: 0021-9258 [Print] United States
• PMID: 15111623 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Amino Acids
• Antigens, Surface
• Carrier Proteins
• Glycoproteins
• Interleukin-8
• LY96 protein, human
• Lipopolysaccharides
• Lymphocyte Antigen 96
• Membrane Glycoproteins
• NPC2 protein, human
• Peptides
• Receptors, Cell Surface
• Recombinant Proteins
• TLR4 protein, human
• Toll-Like Receptor 4
• Toll-Like Receptors
• Tumor Necrosis Factor-alpha
• Vesicular Transport Proteins
• Pancreatic Elastase
• cholesterol-binding protein

Topics

• Amino Acid Motifs
• Amino Acid Sequence
• Amino Acids (chemistry)
• Animals
• Antigens, Surface (chemistry)
• Carrier Proteins (chemistry)
• Cell Line
• Circular Dichroism
• Enzyme-Linked Immunosorbent Assay
• Flow Cytometry
• Glycoproteins (chemistry)
• Glycosylation
• Humans
• Interleukin-8 (metabolism)
• Lipopolysaccharides (chemistry)
• Lymphocyte Antigen 96
• Membrane Glycoproteins (chemistry, physiology)
• Mice
• Models, Molecular
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Mutation
• Pancreatic Elastase
• Peptides (chemistry)
• Precipitin Tests
• Protein Binding
• Protein Conformation
• Protein Folding
• Protein Structure, Secondary
• Protein Structure, Tertiary
• Receptors, Cell Surface (chemistry, physiology)
• Recombinant Proteins (chemistry)
• Sequence Homology, Amino Acid
• Signal Transduction
• Toll-Like Receptor 4
• Toll-Like Receptors
• Tumor Necrosis Factor-alpha (metabolism)
• Vesicular Transport Proteins