Interactive sites in the MyD88 toll/interleukin (IL) 1 receptor domain responsible for coupling to the IL1β signaling pathway

Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1 beta, a key inflammatory cytokine. IL1 beta receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1 beta-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1 beta signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1 beta-induced and NF kappa B-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix alpha A, 195-197 in Box 2, and 275 in beta E-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro(200) by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a three-dimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed.