Abstract
IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors.
Highlights
The endoplasmic reticulum is responsible for folding secretory proteins
Our crystal structure shows apo-human IRE1 (hIRE1) is a symmetrical dimer in a back-to-back conformation (Figure 1A) similar to the structure of phosphorylated yeast IRE1 (yIRE1) (Figure 1B), and distinct from the face-to-face dimer previously observed in ADP-bound hIRE (Figure 1C)
This contrasts with the hIRE1-ADP structure determined previously that has a markedly different kinase active site in which the side chain of Tyr628 from β4 points down into the active site, forming hydrogen bonds to the DFG motif (Figure 2C) [22]
Summary
At homeostasis the folding capacity of the ER and the amount of secretory protein synthesis are balanced. If protein synthesis eclipses folding capacity, the accumulation of misfolded protein is sensed and a recovery mechanism, termed the unfolded protein response (UPR), is activated. UPR signaling reduces protein translation and increases the ER lumen volume and protein folding capacity, if homeostasis cannot be restored the UPR signals for the cell to undergo apoptosis [1, 2]. It has an N-terminal stress sensing domain in the ER lumen and a cytoplasmic kinase/ ribonuclease domain which are connected by a single-pass transmembrane helix [4]. IRE1 undergoes autophosphorylation, generating a specific endoribonuclease activity that hydrolyses 2 stem-loops in the pre-mRNA for a bZIP transcription factor [5, 6]. Re-ligation of the RNA results in a frame shift, and subsequently an active transcription factor is translated, HAC1 in yeast and XBP1 in metazoans [7], which increases transcription of UPR target genes [1, 2]
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