Activation mechanisms of the innate immune system structure-function studies of interactions between death domains of MyD88 and IRAK proteins

Resumen

Toll-like receptors (TLRs) mediate evolutionary conserved immune responses against invading microorganisms by recognizing specific pathogen-associated molecules. Major inflammatory pathways elicited upon engagement of TLRs and interleukin-1 receptor (IL-1R) proceed through the bipartite adaptor, MyD88. The adaptor is comprised of an N-terminal death domain (DD, residues Thr17 to Gln120 in the human protein) and a C-terminal Toll¿IL-1R (TIR) domain (Glu159 to Pro296), connected by a long, unstructured linker. TLR¿MyD88 and IL- 1R¿MyD88 complexes bind protein kinases IRAK-1 and 4 to trigger a phosphorylation cascade that eventually culminates with NF-¿B activation and downstream transcription of pro- and antiinflammatory genes. This inflammatory response, in turn, is down-regulated by the catalytically disabled member of the family, IRAK-M, although the mechanisms underlying this process are currently unknown. In addition, although it is known that formation of MyD88¿IRAK complexes depends upon homotypic interactions of their death domains, the architecture of these complexes also remains in dispute. A truncated splice variant of the adaptor, MyD88s, which lacks residues Glu110 to Leu154, cannot recruit IRAK-4 and fails to elicit inflammatory responses. In the present work, with the aim of elucidating the interactions between MyD88 and IRAK proteins in the TLR/IL-1R signaling pathway, we have cloned, overexpressed, purified and characterized recombinant proteins comprising the death domains of MyD88, IRAK-1, IRAK-M and IRAK-4, both alone and extended by the linker to C-terminal TIR or kinase domains, respectively. Using these recombinant fragments, we have shown that the DDs of MyD88 and IRAK proteins form large aggregates in solution. In addition, we have demonstrated that the death domain of the adaptor is able to interact with similar domains from IRAK-M and IRAK-4. Of particular note, both forms of recombinant MyD88 are able to bind to the linker-extended variant of IRAK-4 death domain and pull down native, full-length IRAK-4 from monocyte extracts. Specifically, we show that residues Glu110-Gln120 including the C-terminal helix ¿7 of MyD88, but not the irregular DD-TIR linker, are required for IRAK-4 recruitment, while residues up to Lys115 in the DD¿kinase linker of IRAK-4, and not beyond, are needed for strong interactions with the adaptor. Further, we demonstrate that residues beyond Glu143 in the MyD88 DD-TIR linker, at least, are dispensable for IRAK-M binding to MyD88. Altogether, the current findings can be integrated in a model for sequential assembly of membrane-proximal MyD88 complexes, as we propose, and provide a straightforward explanation for the negative regulation of innate immune responses mediated by MyD88s.