Structure and function of the stress-sensing domain of IRE1 and PERK in the unfolded protein response

The endoplasmic reticulum (ER) is the principal site for the folding and maturation of newly synthesized transmembrane and secretory proteins. Disruption of ER homeostasis leads to accumulation of unfolded proteins and activates an intracellular signal transduction pathway termed the unfolded protein response (UPR). Upon UPR induction, multiple adaptive responses are activated to promote cell survival. In particular, the expression of the ER chaperone BiP is upregulated to increase the folding capacity of the ER. The UPR is transmitted from the ER lumen to the nucleus and the cytoplasm by two proximal sensors that are the protein kinase receptors IRE1 and PERK. The key question in the field is how these protein kinase receptors are turned ‘on’ and ‘off’. In this study we provide a detailed functional, biochemical and structural investigation of the amino-terminal lumenal domains (NLDs) from IRE1 and PERK.; Functional analysis in yeast showed that the NLDs from IRE1 and PERK are responsible for detecting unfolded proteins and they sense this stress signal by a common mechanism in a ligand-independent manner. To investigate the biochemical mechanism for NLD signal transmission, a functional NLD of human IRE1α was produced in a soluble form in mammalian cells. The soluble NLD is a homotypic ligand-independent dimerization domain. It contains three conserved motifs at the N-terminus that are necessary and sufficient for dimerization and signaling the UPR. Biochemical studies demonstrated that hydrophobic interactions and intermolecular disulfide bonding mediate NLD dimerization. However, the hydrophobic interactions are the driving force for dimerization. Furthermore, our studies revealed potential BiP-binding determinants within the NLD. BiP may negatively regulate NLD dimerization and therefore, receptor activation. To investigate the structural basis for the ER transmembrane signaling event, we established a bacterial system to obtain large quantity of purified NLD proteins for structural studies. Both native and selenomethionyl crystals were obtained and diffraction data collected. Preliminary structural analysis revealed an extensive hydrophobic dimer interface composed of four extended β strands from each subunit. Collectively, our results demonstrate that the NLD evolved as a robust functional dimerization module that is necessary to sense unfolded proteins in the ER lumen.