1. Cell Biology

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

  1. Niko Amin-Wetzel
  2. Lisa Neidhardt
  3. Yahui Yan
  4. Matthias P Mayer
  5. David Ron  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. DKFZ-ZMBH Alliance, Germany
https://doi.org/10.7554/eLife.50793
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Cite this article
  1. Niko Amin-Wetzel
  2. Lisa Neidhardt
  3. Yahui Yan
  4. Matthias P Mayer
  5. David Ron
(2019)
Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR
eLife 8:e50793.
https://doi.org/10.7554/eLife.50793
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10 figures, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Fusion of ERdj4’s J-domain to IRE1LD promotes efficient BiP association thereby repressing IRE1 activity in cells.

(A) Two dimensional plots of CHOP::GFP and XBP1s::Turquoise signals from CHO-K1 dual UPR reporter cells stably expressing the indicated IRE1 variants [IRE1 wild-type (wt), J-IRE1 or JQPD-IRE1 fusion; see Figure 1—figure supplement 1A, for schema of the alleles] from the endogenous Ern1 locus untreated and treated with the ER stressor tunicamycin (Tm). Clones used for the analysis were derived from an IRE1 null (ΔIRE1) parental cell line. A representative data set out of three independent experiments is shown. Note the low XBP1::Turquoise intensity in stressed J-IRE1 rescued ΔIRE1 cells. (B) Two dimensional plots of mCherry and XBP1s::Turquoise signals of clones described in ‘A’ transiently transfected with a plasmid encoding the SubA protease, which cleaves BiP at its interdomain linker and an mCherry fluorescent transfection marker. The inactive SubAS272A mutant was used as control. Representative data from nine biological repeats is shown. (C) Immunoblot (IB) of endogenous IRE1 and associated BiP recovered from the indicated cell lines by immunoprecipitation (IP) of IRE1. Quantification of the ratio of BiP to IRE1 signals in three independent experiments is shown on the right (mean ± standard deviation, n.s.: not significant, *: p<0.05, unpaired parametric Student’s t test). BiP in input cell lysates is provided as loading control. (Figure 1—source data 1) (D) Coomassie-stained SDS-PAGE gel of biotinylated IRE1LD (IRE1LD-bio) and a fusion of ERdj4’s J-domain to IRE1LD (J-IRE1LD-bio, as in ‘A’) and BiP, both recovered on a streptavidin matrix from samples constituted as indicated. Protein concentrations were 5 µM IRE1LD-bio variants, 30 µM BiP, 8 µM ERdj4, and 2 mM ATP. Proteins were eluted in SDS sample buffer. A representative data set out of three independent experiments is shown. (E) Time-dependent change in donor fluorescence of the indicated IRE1LD FRET pair incubated at t = 0 with the components shown to the right. IRE1LD proteins were either labelled with the donor molecule Oregon green 488 (OG) or the acceptor molecule TAMRA (TMR). Protein concentrations were 0.2 µM IRE1LD FRET pair, 30 µM BiP, 2.5 µM ERdj4 and 2 mM ATP. A representative graph of three independent experiments is shown (Figure 1—source data 2).

Figure 1—source data 1

Source data for Figure 1C.

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Figure 1—source data 2

Source data for Figure 1E .

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Figure 1—figure supplement 1
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Modification of the endogenous Ern1 locus to introduce IRE1LD variants.

(A) Schematic description of the homologous recombination platform for creating IRE1-encoding alleles at the endogenous Ern1 locus (ΔIRE1, Kono et al., 2017). Cas9-CRISPR guides generated a large in-frame deletion between exons 2 and 11 in the endogenous Ern1 locus. In the resultant Ern1 null cell line (ΔLD15) the locus can be targeted with a unique Cas9-CRISPR guide and appropriate repair templates containing an intron-free minigene consisting of exons 2–11 to generate IRE1LD variants stably expressed from the endogenous locus by homology-directed repair (HDR). (B) Scheme of IRE1 variants: wild-type IRE1 (wt) and chimeric J-domain IRE1 fusion proteins expressed from the endogenous Ern1 locus to probe the effect of BiP binding to IRE1 activity in cells. The J-domain (or QPD mutant) of ERdj4 was fused N-terminally to IRE1LD, whereby ERdj4’s glycine-phenylalanine-rich (G/F) region was included as a flexible linker between both domains.

Figure 2 with 2 supplements
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Binding of MPZ-N peptide to IRE1LD does not promote IRE1LD dimerisation.

(A) Size-exclusion chromatography (SEC) elution profiles of TAMRA (TMR)-labelled wild-type IRE1LD at the indicated concentrations in presence and absence of MPZ-N peptide. TMR fluorescence is plotted against elution time (see: Figure 2—source data 1). (B) SEC elution profiles (as in ‘A’), but with protein absorbance at 280 nm (A280) plotted against elution time. The inset is a zoom into the segment of the chromatogram encompassing the IRE1LD proteins, whose absorbance is dwarfed by the peak of free peptide (eluting at ~24 min). The heterogenous peaks eluting between 15 and 20 min in the sample loaded with 1 mM MPZ-N peptide, likely reflected peptide oligomerisation (see: Figure 2—source data 1). (C) Cartoon representation of the IRE1LD dimer (PDB: 2HZ6) is shown on the left with coloured secondary structures (cyan for helices, red for sheets and magenta for loops). The Gln105 side chain is shown as sticks, a closer view of which is shown on the top right. The bottom right panel shows a similar view of the Gln105Cys mutant (crystallised here), which forms a disulphide bond, covered with clear electron density (black mesh represents the 2mFo − DFc map, contoured at 1.0 σ, including density within 2 Å of the cysteine residues). (D) Anisotropy of FAM labelled MPZ-N peptide (100 nM) in presence of increasing concentrations of either wild-type IRE1LD or disulphide-linked dimeric IRE1Q105C SS. Shown are data from three independent experiments (mean ± SD). Curve fitting was performed in Prism GraphPad 7.0 using Equation 2 in Materials and methods (see: Figure 2—source data 2).

Figure 2—source data 1

Source data for Figure 2A and B.

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Figure 2—source data 2

Source data for Figure 2D.

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Figure 2—figure supplement 1
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Biochemical properties of disulphide-linked dimeric IRE1LD Q105C SS and monomeric variants used to study MPZ-N’s interaction with IRE1LD.

(A) Size-exclusion chromatography (SEC) elution profiles of wild-type (wt) and monomeric IRE1LD W125A and IRE1LD P108A proteins at the indicated concentrations. Protein absorbance at 280 nm (A280) is plotted against elution time. Note the delayed peak elution times of the IRE1LD monomeric variants. The observation that IRE1LD wt and IRE1LD W125A variants eluted in a single peak yet shifting to later elution times with decreasing protein concentrations suggests that these proteins existed in a monomer-dimer exchange regime that is faster than the time scale of SEC. By contrast, IRE1LD P108A was monomeric at all tested concentrations. (B) Cartoon representation of the IRE1LD dimer interface with protomers in light and dark grey (PDB: 2HZ6). Residues that were mutated to create the monomeric variants IRE1LD W125A and IRE1LD P108A are located in close proximity to the dimer interface and are shown in orange or pink sticks, respectively. (C) Coomassie stained non-reducing SDS-PAGE gel of wt IRE1LD and disulphide-linked IRE1LD Q105C SS. (D) Scatter plot of the melting temperature (Tm) of the indicated IRE1LD proteins (at 5 µM) in absence or presence of the reducing agent TCEP or increasing concentrations of MPZ-N peptide measured by differential scanning fluorimetry (DSF) is depicted on the left. Shown are the data points of three independent experiments [mean ± standard deviation (SD)] with monomeric IRE1LD W125A and IRE1LD P108A, wt IRE1LD and a disulphide-linked dimeric IRE1LD Q105C SS or its reduced version IRE1LD Q105C SH. Statistical analysis for indicated conditions was performed in comparison to wt IRE1LD (***: p<0.001, ****: p<0.0001, ordinary one-way ANOVA). Traces of melt curves with their negative first derivatives from a representative experiment are shown on the right (peak fluorescence was normalised). (E) Overlay of the cartoon representation of IRE1LD (PDB: 2HZ6) and the disulphide-linked IRE1LD Q105C SS. The root-mean squared deviation (RMSD) over 227 Cα atoms is 0.46 Å.

Figure 2—figure supplement 1—source data 1

Source data for Figure 2—figure supplement 1A.

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Figure 2—figure supplement 1—source data 2

Source data for Figure 2—figure supplement 1D.

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Figure 2—figure supplement 2
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Implications of the distance constraint arising from the paramagnetic relaxation enhancement (PRE) experiments with IRE1LD and an MPZ-proxyl-labelled peptide (Karagöz et al., 2017) to the possible modes of peptide binding.

(A) Cartoon representation of the IRE1LD dimer with protomers in light and dark grey (PDB: 2HZ6). The two helices flanking the MHC-like groove are coloured in blue. The isoleucines (Ile) whose peaks were suppressed or broadened upon introduction of the spin-labeled MPZ peptide are highlighted in red and yellow, respectively (from Figure 5 in Karagöz et al., 2017, doi.org/10.7554/eLife.30700.016). The incorporated spin label causes broadening of Ile peaks in a range of 10 to 25 Å or entirely erases them within distances of <10 Å (Gottstein et al., 2012). Therefore, the broadening of peaks observed in the NMR spectra of IRE1LD serves as a distance constraint, positioning the labelled residue of the MPZ peptide within 10 Å of Ile186 (in red). The blue mesh of 10 Å diameter centred on Ile186 depicts the distance constrain on the proxyl-labelled Cys5 of the MPZ peptide (LIRYCWLRRQAALQRRISAME). (B). As in ‘A’, but with two extra spheres centred at Ile186 with diameters of 26 Å (yellow meshes) and 58 Å (grey meshes) to show the possible location of the MPZ peptide in either helical or extended conformation, respectively. Also shown is the 8-residues-long peptide (magenta) present in the yeast IRE1LD crystal structure (Credle et al., 2005, PDB: 2BE1, superimposed onto the human model) and a superimposed peptide (cyan) found in the PERKLD structure (Wang et al., 2018, PDB: 5V1D). Note: There are 16 residues between Cys5 and the C-terminus of the MPZ peptide. If extended, the peptide is free to sample a sphere with a radius of ~48 Å from Cys5. Given that the PRE experiments place Cys5 of the peptide anywhere within 10 Å of IRE1LD Ile186, if extended, the peptide is free to explore the entire space encompassed by the grey mesh. Even if constrained to assume a compressed helical conformation, the peptide could be found anywhere within the yellow mesh. The numerous perturbations to the NMR spectrum arising from the presence of unlabelled peptide may reflect either the consequences of a peptide-IRE1LD interaction or an induced conformational change in the IRE1LD (as noted by Karagöz et al., 2017). Therefore, they do not constrain the location of the peptide. Constraint is provided by the PRE experiments, noted above, but these are not incompatible with the peptide binding outside the MHC-like groove.

Figure 3 with 1 supplement
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Identification of flexible regions in IRE1LD that are important for the regulation of IRE1 activity in cells.

(A) Left panel shows a bar diagram of the percentage of amide hydrogen exchange (%ex) of the indicated by IRE1LD segments after 30 and 300 s incubation in D2O. The amino acids (aa) covered by the peptic fragments are indicated on the left. Exchange was corrected for back exchange using a fully deuterated IRE1LD preparation. Protein concentration was 5 µM. Shown are the data of three independent experiments (mean ± standard deviation). Right panel shows a cartoon of the IRE1LD dimer (PDB: 2HZ6) with the left protomer coloured according to %ex at 30 s (areas with no sequence coverage are uncoloured). The location of the putative loop (residues 308–357) and the tail (residues 390–444) are schematically represented as dotted lines (see: Figure 3—source data 1) (B) Schematic description of a directed in vivo CRISPR-Cas9 mutagenesis strategy to probe regions of IRE1LD for their relevance to regulating activity in CHO-K1 cells. Cas9 guides (red triangles) targeted sites across the Ern1 genomic locus encoding the protein’s region of interest. Transfection of individual or pairs of guides resulted in a collection of mutations (insertions and deletions, depicted as blue and red lines). Cell harbouring rare de-repressing mutations of IRE1 (blue) were selected by fluorescence-activated cell sorting (FACS) gated on XBP1s::Turquoise high and CHOP::GFP low signals. The resultant clones were isolated and genotyped. (C) Left panel is a histogram of XBP1s::Turquoise intensity of CHO-K1 dual UPR reporter cell populations transfected with guide-Cas9 encoding plasmids targeting a putative unstructured loop (aa 308–357) within IRE1LD (identified in ‘A’). XBP1s::Turquoise bright cells within population 0 were collected by FACS (FACS1) yielding population 1, followed by a second round of enrichment for bright cells (FACS2 yielding population 2). Population 2 was treated with the IRE1 inhibitor 4µ8c to select against clones exhibiting IRE1-independent reporter activity. The final population was genotypically analysed (representative sequences are shown on the right). Frameshift mutations are coloured in blue and Cas9 cut sites are indicated below.

Figure 3—source data 1

Source data for Figure 3A.

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Figure 3—figure supplement 1
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IRE1’s tail region is involved in maintaining the repressed state of IRE1 in vivo.

(A) Schematic representation of IRE1. The signal peptide (SP), luminal domain (LD), comprised of a structured core (CLD) and an unstructured tail, the transmembrane (TM) domain and the cytosolic effector domains are indicated with their corresponding amino acid (aa) resides below. (B) Representative sequences of clones that were selected as described in Figure 3C after transfection of cells with guide-Cas9 encoding plasmids targeting IRE1’s tail (residues 368–444). Frameshift mutations are coloured in blue and Cas9 cut sites are indicated by arrowheads below.

Figure 4 with 1 supplement
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Cells expressing IRE1LD deletion variants exhibit a de-repressed IRE1 phenotype that correlates with less BiP bound to IRE1.

(A) Bar diagram of median XBP1::Turquoise and CHOP::GFP signals from untreated and tunicamycin (Tm)-treated CHO-K1 dual UPR reporter cells with Ern1 alleles encoding wild-type (wt) or the indicated deletion variants of IRE1 (∆loop, missing residues 313–338, ∆tail, missing residues 391–444, or ∆∆, missing both). Data from four independent experiments is shown [mean ± standard deviation (SD), **: p<0.01, ****: p<0.0001, one-way ANOVA with Sidak’s multiple comparison test] (Figure 4—source data 1). (B) Representative immunoblot (IB) of endogenously-expressed wt or the IRE1 ∆∆ deletion mutant (see ‘A’) and associated BiP recovered by immunoprecipitation (IP) of IRE1. BiP in input cell lysates is provided as a loading control. Quantification of the ratio of BiP to IRE1 signals after IP of three independent experiments is shown below (mean ± SD, *: p<0.05, ratio paired parametric Student’s t test) (see: Figure 4—source data 2) (C) Left panel shows a representative immunoblot of the indicated IRE1 variants with glutathione S-transferase (GST) replacing the cytosolic domain. The proteins were introduced into CHO-K1 cells by transient transfection, and the associated endogenous BiP recovered by glutathione pull down. BiP in input cell lysates is provided as a loading control. Quantification of the ratio of BiP to IRE1 signals in the IP of three independent experiments is shown to the right (mean ± SD, *: p<0.05, **: p<0.01, ***: p<0.001, one-way ANOVA with Sidak’s multiple comparison test).

Figure 4—source data 1

Source data for Figure 4A.

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Figure 4—source data 2

Source data for Figure 4B.

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Figure 4—figure supplement 1
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Single or double deletion of a flexible loop and the tail within IRE1LD de-repressed IRE1 basal activity.

(A) Two dimensional contour plots of untreated and tunicamycin (Tm)-treated CHO-K1 CHOP::GFP and XBP1s::Turquoise dual UPR reporter cells expressing the indicated alleles (as in Figure 4A) analysed by flow cytometry. The measurements were performed after transient transfection of Cas9-CRISPR guides together with the respective repair templates yielding populations that had and had not undergone homologous recombination (‘Analysis of mixed population’). Representative data from four independent experiments is shown. (B) Contour plot (as in ‘A’) of clonal cell lines expressing the same alleles used in the experiment shown in Figure 4B. Representative data from three biological repeats is shown.

Figure 5 with 1 supplement
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Impaired BiP binding and monomerisation of IRE1LD ΔΔ in vitro.

(A) Left panel shows Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [a fusion of ERdj4’s J-domain to IRE1LD wild-type (wt) or ∆∆, as in Figure 1D] and exposed sequentially to the indicated solutions of analyte (containing wt BiP or the client-binding mutant BiPV461F). A representative experiment of three independent repetitions is shown. The traces were subtracted against a background derived from a BLI sensor with no ligand and the BLI signals (displacement) were set to zero after the first washing step. Quantification of the dissociation rate constants koff after association in presence of 0.15 µM BiP and 2 mM ATP are shown to the right. Traces were fitted to a two-phase dissociation function in Prism GraphPad 7.0. Shown are the mean ± standard deviation (SD) of three independent repetitions (n.s.: not significant, unpaired parametric Student’s t test). (B) Coomassie-stained SDS-PAGE gel of biotinylated wt IRE1LD (IRE1LD-bio), double deleted IRE1LD ∆∆ (IRE1LD ∆∆-bio) and BiP, recovered on a streptavidin matrix from samples constituted as indicated. 2 mM ATP was used during wash steps of the matrix when indicated. A representative data set is shown. Quantification of the ratio of BiP to IRE1 signals in the relevant samples after pull down from three independent experiments is shown on the right (mean ± SD, *: p<0.05, **: p<0.01, one-way ANOVA with Sidak’s multiple comparison test). (C) Time-dependent change in donor fluorescence of the indicated IRE1LD FRET pair incubated at t = 0 with the components shown to the right. The asterisks mark samples set up with a mock FRET sensor lacking the IRE1LD donor-labelled molecule. Protein concentrations were 0.2 µM FRET pair, 30 µM BiP, 2.5 µM full-length ERdj4 (or its isolated J-domain) and 2 mM ATP. A representative experiment of three independent repetitions is shown. When indicated, the data points were fitted to a one-phase association function in Prism GraphPad 7.0; the initial velocity represents the slope of the curve at time point zero (mean ± SD, ***: p<0.001, unpaired parametric Student’s t test) (see: Figure 5—source data 1).

Figure 5—source data 1

Source data for Figure 5A.

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Figure 5—source data 2

Source data for Figure 5C.

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Figure 5—figure supplement 1
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The ∆∆ deletion does not affect the stability of the IRE1LD dimer.

(A) Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [wild-type (wt) or IRE1LD ∆∆] and exposed sequentially to the indicated solutions of analyte. 2 µM BiP, 6.8 µM full-length ERdj4, or isolated J-domain and 2 mM ATP. A representative experiment of three independent repetitions is shown (processed as in Figure 5A). (B) As in Figure 5A but comparing the monomeric J-IRE1LD P108A and the disulphide-linked J-IRE1LD Q105C SS proteins as ligands on the sensor. A representative experiment of three independent repetitions is shown. (C) Size-exclusion chromatography (SEC) elution profiles of TAMRA (TMR)-labelled wt and IRE1LD ΔΔ proteins at the indicated concentrations. TMR fluorescence is plotted against elution time [note: fluorescent labelling was used to detect a signal at the low protein concentration (conc.) required to generate a pool of monomeric IRE1LD] (see: Figure 5—source data 2) (D) Plot of peak elution time points derived from ‘C’ against the protein concentration on a logarithmic scale for IRE1LD and IRE1LD ΔΔ. Curve fitting was performed in Prism GraphPad 7.0 using a sigmoidal function and calculated K1/2 max values are displayed underneath the curves.

Figure 5—figure supplement 1—source data 1

Source data for Figure 5—figure supplement 1B.

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Figure 5—figure supplement 1—source data 2

Source data for Figure 5—figure supplement 1C.

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Figure 6 with 1 supplement
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BiP-mediated monomerisation of IRE1LD ∆∆ assessed by hydrogen exchange mass spectrometry (HX-MS).

(A) Difference plot of deuteron incorporation comparing wild-type (wt) IRE1LD with the monomeric mutants IRE1LD W125A (orange trace) or IRE1LD P108A (pink trace) after 30 s incubation in D2O [see Table 2 for the amino acid (aa) sequence of the individual segments]. Protein concentration was 5 µM. Shown are data from three independent experiments [mean ± standard deviation (SD)]. Boxes 1 and 2 highlight regions of greater hydrogen exchange (HX) in the monomeric mutants compared to wt IRE1LD that were analysed in presence of chaperones in ‘C’ (see Figure 6—source data 1). (B) Cartoon representation of the IRE1LD dimer (PDB: 2HZ6) coloured according to the difference of deuteron incorporation between wt and IRE1LD P108A after 30 s of incubation in D2O (from ‘A’). (C) Difference plot of the deuteron incorporation between the untreated sample and samples exposed to the indicated additives. The data for the same peptic peptides from wt IRE1LD and the IRE1LD ΔΔ mutant are displayed separately. Protein concentrations were 5 µM IRE1LD (wt or ∆∆ mutant), 30 µM BiP (wt or V461F mutant), 6 µM ERdj4 (wt or QPD mutant) and 2 mM ATP. Shown are the means ± SD of three data sets acquired after 30 s incubation in D2O (the corresponding 300 s data set is presented in Figure 6—figure supplement 1A and C) (see Figure 6—source data 2).

Figure 6—source data 1

Source data for Figure 6A and Figure 6—figure supplement 1A.

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Figure 6—source data 2

Source data for Figure 6C and Figure 6—figure supplement 1C.

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Figure 6—figure supplement 1
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HX-MS evidence for impaired BiP- and ERdj4-driven monomerisation of IRE1LD ΔΔ.

(A) Difference plot of deuteron incorporation into the indicated peptic peptides of wild-type (wt) versus IRE1LD W125A or wt versus IRE1LD P108A after 300 s incubation in D2O (as in Figure 6A) (see Figure 6—source data 1). (B) Overlay of representative spectra showing the isotope cluster of peptic fragment 779.0873+ (residues 86–106) from wt or IRE1LD ∆∆, untreated (black) or exposed to BiP, ERdj4 and ATP (red) after 300 s incubation in D2O. Peaks arising from overlapping BiP peptides are coloured in grey. Note the stronger shift of the peaks towards higher m/z values (indicating an increased deuteron incorporation) in case of wt IRE1LD in presence of BiP, ERdj4 and ATP. (C) Difference plot of the deuteron incorporation between the untreated sample and samples exposed to the indicated additives. The data for the same peptic peptides from wt and the IRE1LD ΔΔ mutant are displayed separately (as in Figure 6C, but after 300 s incubation in D2O) (see Figure 6—source data 2) (D) Difference plot of deuteron incorporation into the indicated peptic peptides of wt versus IRE1LD ∆∆ mutant after 30 or 300 s incubation in D2O as in Figure 6A (in absence of chaperones). Regions deleted in IRE1LD ΔΔ are excluded from the difference plot.

Figure 6—figure supplement 1—source data 1

Source data for Figure 6—figure supplement 1E.

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Figure 7 with 1 supplement
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Analysis of bimodally-distributed isotope clusters of IRE1LD peptic peptides reveals active destabilisation of the IRE1LD dimer by BiP.

(A) Intensity distributions of the isotope clusters of peptide 655.273+ (residues 297–302) from IRE1LD, untreated or exposed to BiP, ERDj4 and ATP (30 min at 30°C) following different incubation times in D2O, as indicated. Curves are fits of the sum of two Gaussian distributions (Prism GraphPad 7.0, see Equation 4 in Materials and methods). A representative plot of three independent experiments is shown. (see: Figure 7—source data 1) (B) Plot of time-dependent change in the fractional contribution of high mass species to the isotope clusters of peptide 655.273+ (from ‘A’) calculated as described in Figure 7—figure supplement 1A and B. Shown are data points from three independent samples of IRE1LD in presence and absence of BiP, ERdj4 and ATP. The curves were fitted to a one-phase association model in Prism GraphPad 7.0. Data for a second informative peptide is shown in Figure 7—figure supplement 1C. (C) Bar diagram of the transition rate constant ktrans extracted by analysis of bimodal distributions in the isotope clusters of peptic fragments 636.3802+ and 655.273+ from IRE1LD in presence and absence of BiP, ERdj4 and ATP (from Figure 7B and Figure 7—figure supplement 1C). All the data points from three independent experiments are shown and the mean ± standard deviation (**: p<0.01, ****: p<0.0001, one-way ANOVA with Sidak’s multiple comparison test).

Figure 7—source data 1

Source data for Figure 7A and B and Figure 7—figure supplement 1A, B, C.

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Figure 7—figure supplement 1
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Analysis of the bimodal distributions of the isotope clusters detected by HX-MS.

(A) Shown are original spectra of peptic peptide 655.273+ (residues 297–302) from monomeric IRE1LD P108A (left panel) or wild-type IRE1LD (right panel) at the indicated time point of incubation in D2O. The contribution of the low (blue) and high (red) exchanging populations to the peaks was extracted by fitting the data to a two Gaussian distribution model (Equation 4). Unexchanged and 100% exchanged spectra are provided as references. (B) Plot of the two individual Gaussian distributions of the high (h) and low (l) exchanging populations deconvoluted computationally in Prism GraphPad 7.0 from the composite data set shown in Figure 7A for each time point of incubation in D2O. (C) Plot of time-dependent change in the fractional contribution of high mass species to the isotope clusters of peptic peptide 636.3802+ (residues 96–106). Shown are data points from three independent samples of IRE1LD in presence and absence of BiP, ERdj4 and ATP (as in Figure 7B). The curves were fitted to a one-phase association model in Prism GraphPad 7.0.

Figure 8
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Cartoon depicting features of BiP-mediated regulation of IRE1 activity.

In stressed cells unfolded proteins compete for BiP, exposing IRE1LD to a default dimeric active state, specified by the kinetics of the monomer-dimer equilibrium (left panel). In compensated cells BiP (assisted by ERdj4 and possibly other J-domain proteins, not shown) binds flexible regions of IRE1LD. Engagement of these regions in the IRE1LD dimer may favor active dimer disassembly by entropic pulling or allosterically induced conformational changes (right panel). BiP binding to the same flexible regions of the IRE1LD monomer, may inactivate IRE1 by disfavoring re-dimerisation (right panel). The dynamic nature of BiP binding, which entails cycles for ATP hydrolysis-driven client engagement and nucleotide exchange-mediated release, ensures that IRE1 activity is kinetically coupled to the balance between unfolded protein load and folding capacity of the cell.

Author response image 1
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BiP binding score based on Blond-Elguindi et al. (1993) of hepta-peptides within the entire human IRE1LD’s sequence.

Hepta-peptides having a score of >10 are predicted to have an extremely high probability of binding (black dashed line), peptides with scores between +6 and +10 are predicted to have odds of three to one of having binding activity (grey dashed line), scores between 0 and +5 are predicted to have little predictive value and peptides with scores below 0 are predicted to almost always lack the capacity to bind BiP.

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Bar diagram of median XBP1::Turquoise and CHOP::GFP signals from untreated and tunicamycin (Tm)-treated CHO-K1 dual UPR reporter cells with Ern1 alleles encoding wild type (wt) or the indicated mutant variants of IRE1 (∆loop, missing residues 313-338).

Data from two independent experiments is shown (means ± range)..

Tables

Table 1
Data collection and refinement statistics of IRE1LDQ105C SS.
Data collection
Synchrotron stationsDls i04-1
Space groupP6522
a,b,c; Å182.77, 182.77, 68.45
α, β, γ; ⁰90.00, 90.00, 120.00
Resolution, Å91.39–3.55 (3.89–3.55)*
Rmerge0.180 (2.242)*
I/σ(I)11.7 (1.5)*
CC1/21.000 (0.797)*
No. of unique reflections8590 (1996)*
Completeness, %100.0 (100.0)*
Redundancy19.3 (19.9)*
Refinement
Rwork/Rfree0.323/0.332
No. of atoms (non H)1784
Average B-factors127
RMS Bond lengths Å0.003
RMS Bond angles,⁰0.606
Ramachandran favoured region, %95.85
Ramachandran outliers, %0
MolProbity score†1.51 (100th)
PDB code6SHC

  1. * Values in parentheses are for highest-resolution shell.

    † 100† 100th percentile is the best among structures of comparable resolutions. 0th percentile is the worst.

Table 2
List of IRE1LD peptic peptides analysed by hydrogen-1H/2H-exchange mass spectrometry (HX-MS) containing the respective m/z values, charge (z) and sequence of each peptide.

Note that the N-terminal amide hydrogen of each peptic fragment exchanges too fast to be detectable with this method. Hence, the N-terminal residue was excluded from the data analysis.

ResiduesM/zZSequence
24–36631.3452STSTVTLPETLL
37–45938.4781FVSTLDGSL
46–59396.7304HAVSKRTGSIKWTL
77–85927.4351LPDPNDGSL
86–106779.0873YTLGSKNNEGLTKLPFTIPEL
96–106636.3802LTKLPFTIPEL
107–1191316.6801VQASPSRSSDGIL
120–128390.1993YMGKKQDIW
130–134735.4241YVIDLL
134–145631.8322LTGEKQQTLSSA
147–1571090.5631ADSLSPSTSLL
157–168730.8742LYLGRTEYTITM
168–175522.2422MYDTKTRE
176–183535.7722LRWNATYF
186–1951031.4471AASLPEDDVD
196–208727.8372YKMSHFVSNGDGL
209–221703.3432VVTVDSESGDVLW
221–232697.8562WIQNYASPVVAF
233–2401050.5371YVWQREGL
241–248332.8643RKVMHINV
253–258406.7352LRYLTF
280–287444.282KSKLTPTL
288–2961017.5251YVGKYSTSL
297–302655.2731YASPSM
303–316474.2683VHEGVAVVPRGSTL
317–335956.9782PLLEGPQTDGVTIGDKGES
343–360534.3074VKFDPGLKSKNKLNYLRN
365–378503.5883IGHHETPLSASTKM
379–404516.6066LERFPNNLPKHRENVIPADSEKKSFE
410–424810.3762VDQTSENAPTTVSRD
410–443727.1495VDQTSENAPTTVSRDVEEKPAHAPARPEAPVDSM
Key resources table
Reagent type
(species) or resource		DesignationSource or
reference		IdentifiersAdditional
information
Strain, strain background (Escherichia coli)BL21 C3013 E. coliNEBCat no: C3013I
Strain, strain background (Escherichia coli)Origami B(DE3) E. coliNovagen/MERCKCat no: 70837
AntibodyAnti-mouse IRE1α serum (rabbit polyclonal)Bertolotti et al., 2000NY200used at 1/1000
Antibodyanti-hamster BiP (chicken polyclonal)Avezov et al., 2013Anti-BiPused at 1/1000
AntibodyAnti-GST (polyclonal rabbit)Ron and Habener, 1992Anti-CHOPused at 1/1000
Cell line, (Cricetulus griseus)Clone S21 a derivative of RRID: CVCL_0214Sekine et al., 2016CHO-K1 S21CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter cell line
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise ∆LD 15Kono et al., 2017∆IRE1CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise IRE1 wild-typeThis paperIRE1 wild-typeCHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line reconstituted with IRE1 wild-type
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise IRE1 ∆∆This paperIRE1 ∆∆CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line reconstituted with IRE1 ∆∆ (missing residues 313–338 and 391–444)
Peptide, recombinant proteinMPZ-NKaragöz et al., 2017MPZ-N12-mer peptide (MPZ-N) derived from myelin protein zero
Peptide, recombinant proteinFAM-MPZ-NKaragöz et al., 2017FAM-MPZ-NFAM labelled 12-mer peptide (MPZ-N) derived from myelin protein zero
Software, algorithmPrismGraphPad
Software, algorithmFlowJo,LLC,
Software, algorithmData Analysis 4.1Bruker
Chemical compound, drugTunicamycinMelfordCat no: T2250
Chemical compound, drug2-DeoxyglucoseSigmaCat no: D6134
Chemical compound, drug4μ8cTocris BioscienceCat no: 4479
Chemical compound, drugDigitoninCalbiochemCat no: 300410
Chemical compound, drugBiotin-NHS esterSigmaCat no: H1759
Chemical compound, drugProtease inhibitorsSigma Aldrich (MERCK)S8830
Chemical compound, drugOregon Green-iodoacetic acidThermoFisherCat no: O6010
Chemical compound, drugTAMRA-maleimideSigmaCat no: 94506
Chemical compound, drugPhosphocreatineSigmaCat no: 10621714001
Chemical compound, drugCreatine kinaseSigmaCat no: C3755
Recombinant DNA reagenthaBiP_27–654_pQE10 (plasmid)Petrova et al., 2008UK173N-terminally His6-tagged hamster BiP
Recombinant DNA reagenthaBiP_27–654_V461F_pQE10 (plasmid)Petrova et al., 2008UK182N-terminally His6-tagged hamster BiP V461F
Recombinant DNA reagenthaBiP_27–654_ADDA_pQE10 (plasmid)Preissler et al., 2015aUK984N-terminally His6-tagged hamster BiP ADDA
Recombinant DNA reagentH6_Ulp1_pET28b (plasmid)This studyUK1249H6-tagged Ulp1
Recombinant DNA reagentpCEFL_mCherry_3XFLAG_C (plasmid)Sekine et al., 2016UK1314pCEFL with 3XFLAG_C tagged from mCherry-tagged plasmid
Recombinant DNA reagentBPPTSP_SubA_22–347_3XFLAG_KDEL_pUC57_Acc65I_based_pCEFL_mCherry (plasmid)This studyUK14523xFLAG-tagged SubA with KDEL on mCherry-tagged plasmid
Recombinant DNA reagentBPPTSP_SubA_22–347_S272A_3XFLAG_KDEL_pUC57_Acc65I_based_pCEFL_mCherry (plasmid)This studyUK14593xFLAG-tagged SubAS272Awith KDEL mCherry-tagged plasmid
Recombinant DNA reagenthIRE1_19–486_dC_GST_del3UTR _pCDNA3 (plasmid)Amin-Wetzel et al., 2017UK1703C-GST-tagged cysteine-free human IRE1
Recombinant DNA reagentCHO_IRE1_guideC15.1_pSpCas9(BB)−2A-mCherry (plasmid)Kono et al., 2017UK1903Cas9 and guide targeting IRE1 in CHO-K1 ∆LD clone 15 (mCherry-tagged)
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_reptemp4_pCR-Blunt2-TOPO (plasmid)Kono et al., 2017UK1968Repair template for wild-type IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentSmt3_cgERdj4_24–222_pET-21a (plasmid)Amin-Wetzel et al., 2017UK2012N-Smt3-tagged Chinese amster ERdj4 24–222
Recombinant DNA reagentSmt3_J4_domain_24–90_pET-21a (plasmid)Amin-Wetzel et al., 2017UK2041N-Smt3-tagged Chinese hamster ERdj4 24–90
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 (plasmid)This studyUK2042N-His6-Smt3-tagged wild-type human IRE1LD24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 Q105C (plasmid)Amin-Wetzel et al., 2017UK2045N-His6-Smt3-tagged cysteine-free human IRE1LD Q105C24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 R234C (plasmid)Amin-Wetzel et al., 2017UK2048N-His6-Smt3-tagged cysteine-free human IRE1LD24–444, R234C (FRET probe)
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 S112C (plasmid)Amin-Wetzel et al., 2017UK2076N-His6-Smt3-tagged cysteine-free human IRE1LD24–444, S112C (FRET probe)
Recombinant DNA reagentSmt3_cgERdj4_24–222_GS6_MalE_pET21a (plasmid)Amin-Wetzel et al., 2017UK2108N-Smt3-ERdj4-MBP Chinese hamster 24–222
Recombinant DNA reagentSmt3_cgERdj4_24–222_QPD_GS6_MalE_pET21a (plasmid)Amin-Wetzel et al., 2017UK2119N-Smt3-ERdj4-MBP Chinese hamster residues 24–222 H54Q
Recombinant DNA reagentIRE1a_LD_∆C_24–443_AviTag_H6_pET30a (plasmid)This studyUK2246C-Avi-His6-tagged cysteine-free human IRE1LD24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_Q105C_24_390 (plasmid)This studyUK2304N-His6-Smt3-tagged cysteine-free human IRE1LD Q105C24–390
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_390_∆313–338_S112C (plasmid)This studyUK2370N-His6-Smt3-tagged cysteine-free human IRE1LD ∆∆ (313-338, 391-444) S112C, FRET probe
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d313-338_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2384Repair template for IRE1 ∆loop (d313-338) reconstitution in CHO-K1 cells
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d391-444_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2385Repair template for IRE1 ∆tail (d391-444) reconstitution in CHO-K1 cells
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d313-338_d391-440_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2386Repair template for IRE1 ∆∆ (d313-338, 391–444) reconstitution in CHO-K1 cells
Recombinant DNA reagenthIRE1α_19–486_dC_ d313-338_d391-440_GST_del3UTR _pCDNA3 (plasmid)This studyUK2401C-GST-tagged cysteine-free human IRE1 ∆∆ (missing residues 313–338 and 391–444)
Recombinant DNA reagenthIRE1α_19–486_dC_ d313-338_GST_del3UTR _pCDNA3 (plasmid)This studyUK2404C-GST-tagged cysteine-free human IRE1 ∆loop (missing residues 313–338)
Recombinant DNA reagenthIRE1α_19–486_dC_ d391-440_GST_del3UTR _pCDNA3 (plasmid)This studyUK2406C-GST-tagged cysteine-free human IRE1 ∆∆ (missing residues 391–444)
Recombinant DNA reagentMet_ERdj4_24–120_Ire1a_LD∆C_24–443_AviTag_H6_pET30a (plasmid)This studyUK2408C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_444_P108A (plasmid)This studyUK2410N-His6-Smt3-tagged cysteine-free human IRE1LD P108Amonomeric mutant 24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_444_W125A (plasmid)This studyUK2411N-His6-Smt3-tagged cysteine-free human IRE1LD W125Amonomeric mutant 24–444
Recombinant DNA reagentMet_ERdj4_24–120_Ire1a_LD∆C_24–443_S112C_AviTag_H6_pET30a (plasmid)This studyUK2412C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein, S112C (FRET probe)
Recombinant DNA reagentJ4_WT_IRE1_LD_CHORepairTemplate (plasmid)This studyUK2425Repair template for chimeric J-IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentJ4_QPD_IRE1_LD_CHORepairTemplate_V1 (plasmid)This studyUK2426Repair template for chimeric JQPD-IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentMet_ERdJ4_24–120_Ire1a_LD∆C_24–443_P108A_AviTag_H6_pET30a (plasmid)This studyUK2428C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein containing monomerising mutation P108A
Recombinant DNA reagentMet_ErdJ4_24–120_IRE1a_LD∆C_24–390_∆313–338_AviTag_H6_pET30a (plasmid)This studyUK2458C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD ∆∆protein (313-338, 391-444)
Recombinant DNA reagentIRE1a_LD∆C_24–390_∆313–338_AviTag_H6_pET30a (plasmid)This studyUK2459C-Avi-His6-tagged cysteine-free human IRE1LD ∆∆(d313-338, 391–444)
Recombinant DNA reagentMet_ERdJ4_24–120_Ire1a_LD∆C_24–443_Q105C_AviTag_H6_pET30a (plasmid)This studyUK2558C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein containing mutation Q105C

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  1. Niko Amin-Wetzel
  2. Lisa Neidhardt
  3. Yahui Yan
  4. Matthias P Mayer
  5. David Ron
(2019)
Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR
eLife 8:e50793.
https://doi.org/10.7554/eLife.50793