Computational and Experimental Structural Characterisation of PIDD1 and its binding partners, CRADD, and BUB1B

p53-Induced Death Domain protein 1 (PIDD1) is a protein that forms high molecular weight complexes, referred to as PIDDosomes. A NEMO-PIDDosome participates in sterile inflammation, while a Caspase-2-PIDDosome arising from the interaction of PIDD1 with CASP2 and RIPK1 domain containing Adaptor with Death Domain (CRADD), and Caspase-2, is involved in cell death. Reported to be assembled sequentially, this is facilitated by PIDD1 autocatalysis, which is cleaved into PIDD1-C first and forms the NEMO-PIDDosome triggered by mild DNA damage, and PIDD1-CC afterwards, which forms the Caspase-2-PIDDosome triggered by irreparable DNA damage. Importantly, BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) was reported to prevent Caspase-2-PIDDosome assembly in irradiated and CHEK1 (Checkpoint Kinase 1)-suppressed cells. In this thesis, I focus on the study of the structure that facilitates the interaction of PIDD1 with CRADD, and of the inhibitor of the Caspase-2-PIDDosome BUB1B by performing computational and experimental studies. The computational structure analyses of the individual proteins along with computational docking of the interactions performed throughout this thesis aimed to contribute towards broadening the knowledge of the abovementioned interactions. Result of these studies, computational predictors identified BUB1B as an intrinsically disordered protein, and its docking to the Death Domain (DD) of PIDD1 suggested the 443-460 BUB1B region as responsible for the interaction with PIDD1-DD. Furthermore, a mechanism of activation and deactivation of CRADD by dynamic rearrangements in closed and open conformations was proposed. Finally, the experimentally characterised crystal structure of phosphomimetic Thr788Asp PIDD1-DD allowed to propose a mechanism of PIDD1-CC activation by the dynamic rearrangement from a closed to an open conformation in response to ATM serine/threonine kinase phosphorylation of Thr788 in PIDD1-DD. Providing insight into the singularities of these interactions supposes the stepping stone towards the development of inhibitors of the PIDD1-BUB1B interaction, which could assist in the restoration of apoptosis in response to irradiation in cancer cells.