Mechanistic characterisation of IRE1α's activation cascade and how it is perturbed by cancer-associated mutations

The unfolded protein response is the cell’s reaction to an increased burden on the endoplasmic reticulum’s (ER) protein folding machinery. The most conserved sensor of ER stress is IRE1α, which clusters in response to stress to initiate a cellular signal. IRE1α activation is a complex, multi-step mechanism, triggered by IRE1α’s luminal domain’s (IRE1-LD) response to fluctuating ER stress levels. Currently, the mechanisms of IRE1-LD’s activation and termination are only partially understood, with conflicting models proposed. Using a set of biophysical approaches, IRE1-LD’s complex conformational landscape is characterised in unstressed conditions, upon addition of unfolded protein mimics (representative of ER stress) and the molecular chaperone BiP. The outputs suggest that in the absence of stress IRE1-LD exists in a conformational equilibrium between monomers, homodimers and homooligomers. In a concentration-dependent manner the unfolded protein mimics shift this equilibrium towards increasingly large oligomers, indicative of a proportional response to ER stress levels. Interestingly, these substrate-induced oligomers adopt fibril-like structures, providing a plausible model of the protein’s clustering in vivo. In turn, addition of the molecular chaperone, BiP to these oligomers results in their disassembly, revealing a novel IRE1α-BiP interaction. Notably, this process requires BiP’s chaperone activity and the presence of ATP, reminiscent of Hsp70-assisted clathrin uncoating. Following this, the effects of four IRE1-LD cancer-associated mutations on the activation cascade are characterised, revealing novel allosteric sites that enable tuning of IRE1-LD’s conformational landscape and thus IRE1α activation. Lastly, solution NMR is used to probe the conformation of IRE1-LD’s functionally important intrinsically disordered regions, revealing an allosteric network between key functional sites in the protein: the substrate-binding cleft, dimerisation and oligomerisation interfaces and the C-terminal juxtamembrane linker. Therefore, the research here augments our understanding of IRE1α’s response to stress and identifies allosteric sites in the protein that offer novel potential therapeutic targets