Towards a molecular understanding of Myddosome organization and regulation of IRAK kinase activity

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Innate immune responses are the first line of defence that are triggered following the recognition of danger signals by pattern sensing receptors such as the Toll-like receptors (TLRs) and activation of cytokine receptors such as the interleukin-1 receptor (IL-1R). A first step following the activation of these receptors is the assembly of large multi-protein signalling platforms called Myddosomes. Within these platforms, the IL-1R-associated kinases (IRAKs) are modified covalently and are activated to trigger a signalling cascade that culminates in a transcriptional program that shapes the immune response. Despite being the focus of intense study over the past two decades, our understanding of the organization of Myddosomes, how the IRAK family members are recruited to these complexes and the mechanisms regulating the activity of the IRAKs is still limited. This is because most structural studies to date have investigated components of Myddosomes in isolation. Further, analysis of Myddosomes and innate immune signalling in cells do not provide a clear picture owing to the enormous temporospatial complexities of the system, as well as redundancy and compensatory mechanisms. The overall goal of this proposal is to reconstitute these central hubs of innate immune signalling in vitro to understand the principles governing the organization of Myddosome complexes and to uncover mechanisms of IRAK activation and regulation. To achieve these goals, we will use purified Myddosomes to define the rules governing the assembly of the supramolecular complex. This approach will allow us to systematically evaluate the principles governing Myddosome assembly and to address important basic questions that are still unanswered to date: Is the composition of Myddosomes controlled at the receptor level? Which of the IRAKs can co-exist in a given Myddosome? How does incorporation into higher order complexes regulate IRAK activity and function? We will determine the structures of these fully assembled complexes by cryo-electron microscopy (cryo- EM). We recently solved the crystal structure of the IRAK3 pseudokinase domain, which revealed a novel mechanism by which it may heterodimerize with the autophosphorylated kinase domain of IRAK4. These studies suggest a hypothesis for how this inactive pseudokinase may modulate the activity of IRAK4, a premise we will investigate. Central to determining the structures of Myddosomes will be to capture distinct states of the complex. Here, we will take advantage of the key insights into IRAK interactions that we recently obtained and will employ mutants that mimic the activated state of the complex. We will also use nanobodies that we have engineered to bind to IRAK2 and IRAK3 to stabilize the complexes. Since the Myddosome complex and IRAK family members are at the heart of innate immunity, this work will provide important fundamental insights into how immune responses are activated and reveal how they could be modulated to treat inflammatory diseases such as arthritis, atherosclerosis, systemic lupus erythematosus and psoriasis.