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

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-kappa B 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.