Identification of Binding Sites for Myeloid Differentiation Primary Response Gene 88 (MyD88) and Toll-like Receptor 4 in MyD88 Adapter-like (Mal)

• Published in: The Journal of biological chemistry Vol. 288; no. 17; pp. 12054 - 12066
• Main Authors: Bovijn, Celia, Desmet, Anne-Sophie, Uyttendaele, Isabel, Van Acker, Tim, Tavernier, Jan, Peelman, Frank
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.04.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Humans; Immunology; Innate Immunity; Lipopolysaccharide (LPS); Mal TIRAP; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Models, Biological; Mutation; MyD88; Myeloid Differentiation Factor 88 - chemistry; Myeloid Differentiation Factor 88 - genetics; Myeloid Differentiation Factor 88 - metabolism; Peptide Mapping - methods; Protein Binding; Protein Multimerization - physiology; Receptors, Interleukin-1 - chemistry; Receptors, Interleukin-1 - genetics; Receptors, Interleukin-1 - metabolism; TLR4; Toll IL-1 Receptor (TIR) Domain; Toll Receptors; Toll-Like Receptor 4 - chemistry; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Toll-like Receptors (TLR); Toll-like Receptors (TLR); Mal TIRAP; Toll Receptors; Toll IL-1 Receptor (TIR) Domain; Innate Immunity; MyD88; TLR4; Lipopolysaccharide (LPS)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.415810

Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex. Background: It is unknown how the TIR domain of the adapter Mal binds MyD88 to TLR4. Results: We identified mutations in the Mal TIR domain that affect its interactions and signaling Conclusion: Four binding sites and the AB loop in the Mal TIR domain are required for binding TLR4 and MyD88. Significance: This work provides new insights in TIR-TIR interactions.