The role of the MyD88 intermediate domain in the formation of the myddosome assembly in innate immunity

Abstract

Myeloid differentiation primary response protein-88 (MyD88) and Interleukin-1 receptor associated kinase 4 (IRAK-4) are two proteins that are involved in post-receptor signalling in innate immunity. These two proteins have multiple domains. However, they both share a death domain which they use for interacting with each other through homotypic death domain interactions. Although the death domains are essential for the interaction of these two proteins, the MyD88 intermediate domain is also essential for the interaction with IRAK-4. A splice variant of MyD88 that lacks the intermediate domain fails to recruit IRAK-4 during signalling. Death domains of both MyD88 and IRAK-4 interact with each other in vitro to form an undecameric complex known as the Myddosome complex. This complex has a unique stoichiometry of seven MyD88 death domain molecules to four IRAK-4 death domain molecules. It was reconstituted using MyD88 that had both the death and intermediate domains. However, there was a research group that reconstituted the Myddosome without the MyD88 intermediate domain. Their finding was contrary to existing literature. This project set out to investigate the role of the MyD88 intermediate domain in the assembly of the Myddosome complex. We therefore expressed two variants of MyD88 in addition to the IRAK-4 death domain to reconstitute the Myddosome complex. The two variants were the MyD88 death domain and MyD88 that contained both the death and intermediate domains. MyD88 that lacked the intermediate domain was found to be an oligomer using size exclusion chromatography and dynamic light scattering. However, MyD88 that contained only the death domain was monomeric. MyD88 that contained both the death and intermediate domain was mixed with IRAK-4 death domain to reconstitute the Myddosome complex. As expected, we managed to form the Myddosome complex. The presence of this complex was confirmed by size exclusion chromatography and SDS-PAGE. On the contrary, the MyD88 death domain that lacked the intermediate domain failed to form the Myddosome complex when mixed with IRAK-4 death domain. This finding showed that the MyD88 intermediate domain was essential for the Myddosome assembly. It was also in support of existing literature that MyD88 does not interact with IRAK-4 in the absence of its intermediate domain.