Functional sites for signaling and binding sites for Mal. (A) The NF-κB reporter gene assay of MD2 cotransfected with LPS-induced (1.0 μg/ml) 293-hTLR4A-HA cells. In these graphs, each column indicates relative luciferase activity of stimulated cells over nonstimulated cells. Color code: black bars indicate a significant increased NF-κB activity, compared with the wild-type group. The statistical significance of differences in luciferase activities between wild type and mutants was analyzed using the Dunnett's multiple comparison test. Statistical significance was assumed to be P < 0.05. (B) Results of the functional assays of LPS/TLR4 signaling presented on the 3D structure of the TIR domain of MyD88. The results of the functional assays are mapped onto the molecular surface of the MyD88 TIR domain. The amino acid residues judged to be significant by the luciferase assay are shown in red, while nonsignificant residues are shown in light brown. The conserved motifs of boxes 1–3 (FDA of box1, VLPG of box2, FW of box3) are shown in blue. (C) Assay of binding of Mal TIR domain and MyD88 TIR domain wild type or mutants. Alanine substitutions in Site II (R196A) or Site III (R288A) in MyD88 resulted in reduced interaction with Mal. The double alanine substituted mutant of Site II and Site III caused complete abolition of the interaction with Mal. (D) Cysteine substitution in R196 also caused reduced interaction with Mal. (E) Plots of relative integrated area of NMR signals, which were derived from NMR titration data, in a function of added volume of Mal-TIR into ¹⁵N MyD88-TIR sample. The best-fit lines to the data, assuming a simple 1:1 complex model, are also shown. Apparent dissociation constants calculated are also indicated with standard errors (see Fig. S3). The black box, red circle, blue triangle, and green triangle indicate the dissociation curves of wild-type, R196A, R288A, and R196A-R288A, respectively.

Structural model of signaling complex formed by MyD88 and Mal. The Mal binding sites, Sites II and III residues, are shown in red, and noncritical residues for signaling and Site I residue are shown in light blue. The positions of the previously reported functional residues (P125, S180, and E190) are shown as orange spheres, and the phosphorylation sites in Mal for signaling, Tyr-86, Tyr 109, and Tyr-159, are shown as yellow spheres.

Solution structure of the TIR domain of human MyD88. (A) A representative ribbon drawing of the NMR structure of MyD88, generated with MOLMOL 2K.2. The notation of the secondary structures (βA-βE, αA-αC, and αE) is based on TLR TIR domains. (B and C) A superimposed representation of MyD88-TIR (sky blue) and the crystal structure of the TIR domain of TLR2 (orange). Although the β-sheet cores are similar (rmsd = 0.90 Å), there are differences in some regions.

Direct interactions of MyD88, Mal, TLR4, and IRAK4. (A) Binding assay of the TLR4 TIR domain with the MyD88 TIR domain or Mal TIR domain. GST-TLR4-TIR binds Mal-TIR but not MyD88-TIR. (B) Binding assay of the IRAK4 death domain with the MyD88 TIR domain or death domain. IRAK4-DD binds the MyD88-DD+ID but not MyD88-TIR.