The Myddosome is a large multimeric complex assembled through homotypic interactions between the Death Domains (DDs) of adjacent proteins. It acts as a scaffold bringing downstream signaling elements into proximity with each other. This allows for the subsequent activation of downstream transcription factors, such as IRFs (Interferon Regulatory Factors) and NFκB.

The crystal structure of the Myddosome was published by Lin et al [1], by combining the Death Domains of MyD88, IRAK4 and IRAK2. The complex was shown to form a left-handed helical tower as it assembles in a staggered hexagonal pattern, due to the three characteristic types of homotypic Death Domain (DD:DD) interactions seen in complexes of the Death Domain Superfamily.

The work discussed in this thesis aimed to expand upon our understanding of the Myddosome complex. An initial bioinformatic analysis was carried out to further investigate the different homotypic Death Domain interfaces responsible for Myddosome assembly. The physical and geometric properties of the complex were examined in detail to identify how they contribute towards the complex’s final structure. Additional computational work provides evolutionary context for the development of the Myddosome, while also drawing links to known non- communicable diseases.

A new type of homotypic Death Domain interface was potentially identified, while examining the interactions formed during Myddosome assembly in silico. It appears that this interface is exclusive to members of the IRAK family. Subsequent in vitro work was initiated to verify these observations and future research possibilities were discussed.

Points of further clarification were identified when examining the 3-dimensional Myddosome structure as published by Lin et al. Unit cell contacts caused by the presence of un-cleaved GST purification tags were found to be responsible for structural distortion seen in the crystallographic structure. Additionally, the MyD88 Intermediate Domain was not included in the structure of the Myddosome, despite studies characterising the MyD88 Intermediate Domain as vital for functional Myddosome assembly. Further in vitro biophysical analysis was proposed to both rectify the distortion and to further characterise the interactions involved in Myddosome assembly in the presence of the MyD88 Intermediate Domain.