Structural basis for recognition of unfolded proteins by the ER stress sensor ERN1/IRE1α

IRE1 is an endoplasmic reticulum sensor that recognizes misfolded proteins to activate the unfolded protein response (UPR). We used cholera toxin (CTx), which activates IRE1 in cells, to understand how unfolded proteins are recognized. In vitro, the A1 subunit of CTx (CTxA1) bound IRE1 lumenal domain (IRE1LD). Global unfolding was not required. Instead, IRE1LD recognized a 7-residue motif within a metastable region of CTxA1 that was also found in microbial and host proteins involved in IRE1 activation. Binding mapped to a pocket on IRE1LD normally occupied by a segment of the IRE1 C-terminal flexible loop implicated in IRE1 regulation. Mutation of the recognition motif blocked CTx-induced IRE1 activation in live cells. These findings describe a mechanism for substrate recognition by IRE1 that induces the UPR.