Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
Molecular Discovery Research, GlaxoSmithKline R&D, Stevenage, SG1 2NY, UK.
Sci Rep. 2016 Nov 23;6:37267. doi: 10.1038/srep37267.
Activation 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).
Toll 样受体的激活诱导二聚体的形成，并将死亡结构域（DD）衔接蛋白 MyD88 募集到一个称为 Myddosome 的寡聚受体后复合物中。Myddosome 是炎症和致癌信号的中心，具有分层排列，其中 6-8 个 MyD88 分子与确切的 4 个 IRAK-4 和 4 个 IRAK-2 组装在一起。在这里，我们表明 IRAK-4 中的保守基序（Ser8-X-X-X-Arg12）发生自动磷酸化，并且磷酸化的 DD 无法形成 Myddosomes。此外，在该位置具有磷酸模拟残基 Asp 的突变 DD 在信号转导和 Myddosome 组装中均受到损害。IRAK-4 Arg12 对于 Myddosome 组装和信号转导也是必不可少的，我们提出磷酸化的 Ser8 诱导 N 端环折叠成α-螺旋。这种构象通过磷酸化 Ser8 和 Arg12 之间的静电相互作用得到稳定，并会破坏 IRAK-4 和 MyD88 之间的关键界面。有趣的是，IRAK-2 不保守此基序，并且在 Myddosome 中有一个替代界面，需要 Arg67，该残基在同源物 IRAK-1 和 3（M）中保守。
