Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses

Daiva M. Mattis, Adam S. Chervin, Diana R. Ranoa, Stacy L. Kelley, Richard I Tapping, David M Kranz

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)203-212
Number of pages10
JournalMolecular Immunology
Volume68
Issue number2
DOIs
StatePublished - May 8 2015

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Toll-Like Receptor 4
Lipopolysaccharides
Yeasts
Proteins
Immune System
Antigen-Presenting Cells
Proline
Alanine
Ligands
Mutation

Keywords

  • Lipopolysaccharide
  • MD-2
  • Toll-like receptor-4
  • Yeast display

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

Cite this

Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses. / Mattis, Daiva M.; Chervin, Adam S.; Ranoa, Diana R.; Kelley, Stacy L.; Tapping, Richard I; Kranz, David M.

In: Molecular Immunology, Vol. 68, No. 2, 08.05.2015, p. 203-212.

Research output: Contribution to journalArticle

Mattis, Daiva M. ; Chervin, Adam S. ; Ranoa, Diana R. ; Kelley, Stacy L. ; Tapping, Richard I ; Kranz, David M. / Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses. In: Molecular Immunology. 2015 ; Vol. 68, No. 2. pp. 203-212.
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