Dossang, Anthony C.G.Motshwene, Precious G.Yang, YangSymmons, Martyn F.Bryant, Clare E.Borman, SattyGeorge, JulieWeber, Alexander N.R.Gay, Nicholas J.2017-01-132017-01-132016-11-23Dossang, A.C.G., Motshwene, P.G., Yang, Y., Symmons, M.F., Bryant, C.E., Borman, S., George, J., Weber, A.N.R. & Gay, N.J. The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold. Scientific Reports. 6, 37267; DOI: 10.1038/srep37267 (2016).2045-232210.1038/srep37267http://hdl.handle.net/2263/58502Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6–8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an α-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).en© The Author(s) 2016 This work is licensed under a Creative Commons Attribution 4.0 International License.MyddosomeElectrostatic interactionDeath domain (DD)N-terminal loopMyddosome signalling scaffoldIRAK-4 death domainArticle