The interaction sites of MyD88 with TRAM.

<p>(A) Luciferase reporter gene activities with wild type and mutant types of the MyD88 TIR domain after IL-18 stimulation. The black bars indicate that the residues show significant difference with wild type. (B) The functional assays of IL-18 signaling presented on the 3D structure of the TIR domain of MyD88. 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 non-significant ones 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 to study the binding of the wild type or mutant TRAM TIR domain and MyD88 TIR domain. The representative alanine substitutions at Site II (R196A) or Site III (R288A) in MyD88 caused a reduced interaction with TRAM. The double alanine substituted mutant at Site II and Site III caused the complete abrogation of the interaction with TRAM. (D) Immunoprecipitation assay between MyD88 wild or R196A–R288A mutant, and TRAM.