Abstract
The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1α is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1α dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1α, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix αK, located between the kinase and RNase domains of IRE1α, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1α differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix αK is a conduit for the activation of IRE1α in response to stress.
Highlights
To address how the activation signal is transmitted from the kinase domain of inositol-requiring enzyme 1 α (IRE1α) to its RNase domain, we focused on a mutation that was discovered in a random mutant screen, L827P, that drastically inactivated this stress sensor despite being far removed from either the kinase or the RNase active sites
Following the trans-autophosphorylation that occurs when IRE1α dimerizes, a conformational change is relayed through residues L827–W833 in helix αK (data here and in [28])
Even though helix αK is in the cytosolic portion of IRE1α and not in the luminal domain or in the transmembrane segment, which are each known to sense endoplasmic reticulum (ER) stress, it is necessary for proper activation of IRE1α
Summary
A derivative of HAP1, a near-haploid human cell line, termed HAP1KO, was engineered by CRISPR-Cas editing, to abolish the expression of IRE1α. The input was the crystal structure of apo human IRE1α (Protein Data Bank [PDB]: 5HGI). It was preminimized using the “minimize_with_cst” application in Rosetta. We followed a ΔΔG _monomer application described by Kellogg et al [26] for estimating stability changes in monomeric proteins in response to point mutations This application uses the input structure of the WT protein to generate a structural model of the point mutant. IRDye-conjugated secondary antibodies were from Li-Cor. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1% NP-40, 20 mM iodoacetamide), the protein content was determined as above, and trypsin was added at the indicated final concentration and incubated on ice for 30 min. Each simulation of WT, L827P, and L827F was done in duplicate
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