title: Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses.
abstract: Bacterial lipopolysaccharide (LPS) activates the innate immune system by forming a complex with myeloid differentiation factor 2 (MD-2) and Toll-like receptor 4 (TLR4), which is present on antigen presenting cells. MD-2 plays an essential role in this activation of the innate immune system as a member of the ternary complex, TLR4:MD-2:LPS. With the goal of further understanding the molecular details of the interaction of MD-2 with LPS and TLR4, and possibly toward engineering dominant negative regulators of the MD-2 protein, here we subjected MD-2 to a mutational analysis using yeast display. The approach included generation of site-directed alanine mutants, and ligand-driven selections of MD-2 mutant libraries. Our findings showed that: (1) proline mutations in the F119-K132 loop that binds LPS were strongly selected for enhanced yeast surface stability, (2) there was a preference for positive-charged side chains (R/K) at residue 120 for LPS binding, and negative-charged side chains (D/E) for TLR4 binding, (3) aromatic residues were strongly preferred at F119 and F121 for LPS binding, and (4) an MD-2 mutant (T84N/D101A/S118A/S120D/K122P) exhibited increased binding to TLR4 but decreased binding to LPS. These studies revealed the impact of specific residues and regions of MD-2 on the binding of LPS and TLR4, and they provide a framework for further directed evolution of the MD-2 protein.
authors: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Daiva M
authors: DM
authors: Mattis
authors: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Adam S
authors: AS
authors: Chervin
authors: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
authors: Diana R
authors: DR
authors: Ranoa
authors: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
authors: Stacy L
authors: SL
authors: Kelley
authors: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
authors: Richard I
authors: RI
authors: Tapping
authors: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA. Electronic address: d-kranz@illinois.edu.
authors: David M
authors: DM
authors: Kranz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015870:Gene Expression; D006801:Humans; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D055672:Static Electricity; D013329:Structure-Activity Relationship; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D019151:Peptide Library; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4
keywords: Lipopolysaccharide; MD-2; Toll-like receptor-4; Yeast display