title: Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2.
abstract: Myeloid differentiation factor 2 (MD-2) is an extracellular protein, associated with the ectodomain of TLR4, that plays a critical role in the recognition of bacterial LPS. Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of the LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within the MD-2 binding pocket as the source of functional differences between hMD-2 and mMD-2. Whereas decreased hydrophobicity of residues 61 and 63 in the hMD-2 binding pocket retained the characteristics of wild-type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia;
authors.forename: Jožica
authors.initials: J
authors.lastname: Vašl
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia;
authors.forename: Alja
authors.initials: A
authors.lastname: Oblak
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia;
authors.forename: Tina T
authors.initials: TT
authors.lastname: Peternelj
authors.affiliation: Center for Biological Research, Superior Council for Scientific Research, 28040 Madrid, Spain;
authors.forename: Javier
authors.identifier: http://orcid.org/0000-0001-8495-6568
authors.initials: J
authors.lastname: Klett
authors.affiliation: Center for Biological Research, Superior Council for Scientific Research, 28040 Madrid, Spain;
authors.forename: Sonsoles
authors.identifier: http://orcid.org/0000-0002-7679-0155
authors.initials: S
authors.lastname: Martín-Santamaría
authors.affiliation: Inflammation Program, Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52241; Veterans Affairs Medical Center, Iowa City, IA 52246; and.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
authors.affiliation: Inflammation Program, Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52241;
authors.forename: Jerrold P
authors.identifier: http://orcid.org/0000-0003-1476-4485
authors.initials: JP
authors.lastname: Weiss
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia; Excellent Nuclear Magnetic Resonance-Future Innovation for Sustainable Technologies Center of Excellence, 1000 Ljubljana, Slovenia roman.jerala@ki.si.
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D051197:Toll-Like Receptor 4
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