Reconstructing the TIR Side of the Myddosome: a Paradigm for TIR-TIR Interactions

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC′-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains. •We fully delineate four interaction sites in the MyD88 TIR domain•Three binding sites lead to a symmetrical and an asymmetrical MyD88 TIR homodimer•The symmetrical dimer is regulated by phosphorylation and binding to Mal•MyD88 TIR assembles into a helical oligomer compatible with the Myddosome structure Vyncke et al. fully delineate four interaction sites in the MyD88 TIR domain. Three sites lead to a helical MyD88 TIR complex, regulated by phosphorylation. A symmetrical MyD88 TIR homodimer forms a composite fourth binding site for Mal.