title: Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40.
abstract: Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.
authors.affiliation: Department of Biochemistry, School of Medicine and School of Health Sciences, Sapporo Medical University, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nishitani
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuzawa
authors.forename: Hitomi
authors.initials: H
authors.lastname: Sano
authors.forename: Takeyuki
authors.initials: T
authors.lastname: Shimizu
authors.forename: Norio
authors.initials: N
authors.lastname: Matsushima
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kuroki
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; D018698:Glutamic Acid; D008239:Lysine; D003545:Cysteine