Schematic representation of the structure-function relationships of IRAK-M based on this study. (A) Functional involvement of the different residues of the Death Domain (DD) of IRAK-M on induction of NF-κB, transcription and translation upon overexpression in 293T cells (data derived from Figures 2 and 3). Graded color code (red->pink->white) indicates the impact of the single mutants on NF-κB in 293T cells (from red important to white no effect). The stretch D19-A23 is not indicated to be essential to NF-κB because mutation of the complete stretch (combined D19-P21 and P22-A23 mutation) results in a mutant with normal NF-κB. (B) Schematic representation of the working mechanism of IRAK-M as inhibitor of MyD88 signaling adapted from Zhou et al., in which IRAK-4/IRAK-M interaction induces transcription of other inhibitors via MEKK3 dependent NF-κB and in which IRAK-2 mediated mRNA stability and translation is inhibited by IRAK-M. Based on our mutagenesis studies both W74 as well as R97 in the IRAK-M-DD are pivotal for IRAK-4/IRAK-M mediated NF-κB activity, but R97 is predicted to bind on the other side of IRAK-4 (see Figure 4). This may induce oligomerization of IRAK-4 and IRAK-1 or IRAK-2 activation, or, since both residues are essential for the unique, previously shown MEKK3 dependent IRAK-M mediated NF-κB activation, IRAK-4 may be sandwiched by IRAK-M to come to this pathway as indicated by W74 and R97 interactions. R97 is not predicted to be involved in the interaction of IRAK-M with IRAK-2 for which F18, D19, P22, R70 and W74 are predicted to be involved.