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. 2010 Apr 23;38(2):291-304.
doi: 10.1016/j.molcel.2010.04.001.

Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1

R Luke Wiseman  1 , Yuhong Zhang, Kenneth P K Lee, Heather P Harding, Cole M Haynes, Joshua Price, Frank Sicheri, David Ron
Affiliations

Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1

R Luke Wiseman et al. Mol Cell. .

Abstract

Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.

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Figures

Figure 1
Figure 1. Small molecule screen for modulators of IRE1 RNase activity
A. Fluorescent-based assay for yeast IRE1 (aa 658–1115) RNase activity. A stem loop RNA substrate incorporating an IRE1 endoribonuclease site (cleavage between G3-C4, labeled bases) was modified 5’ with AlexaFluor 647 (AF647) and 3’ with BlackHoleQuencher3 (BHQ). Cleavage alleviates quenching allowing fluorescence. B. Fluorescence timecourse measuring IRE1’s RNase. The activity of IRE1 (1.0 µM) incubated with increasing concentrations of ADP was measured by the cleavage of the fluorescent substrate (25 nM) depicted in Figure 1A. C. Quantification of the ADP-mediated activation of IRE1 RNase shown in Figure 1B. The initial rate of IRE1 RNase activity was plotted against ADP concentration demonstrating an EC50 of ~40 µM. D. Fluorescence timecourse reporting on cleavage of the IRE1 substrate as in Figure 1A (black) or an altered substrate incorporating deoxyguanosine at position 3 (dG3; red), disrupting cleavage at that site. The maximal signal following cleavage of both substrates was determined by incubation with RNase A (squares). E. Bar graphs depicting the activity of IRE1’s (1.0 µM) RNase in the presence of various small molecules (25 µM) from a collection of kinase inhibitors after a 10 minute reaction; the wells containing quercetin (25 µM) and ADP (2 mM) are indicated.
Figure 2
Figure 2. Quercetin potentiates ADP-mediated IRE1 RNase activity
A. Fluorescence timecourse of substrate (25 nM) cleavage by IRE1 (0.5 µM), alone (circles) and in the presence of ADP (2 mM, squares), quercetin (25 µM, diamonds) or both (triangles). B. Autoradiograph showing the cleavage of a 32P-labeled IRE1 substrate (20 nM), of identical sequence to that depicted in Figure 1A, by IRE1 (0.5 µM) incubated with ADP (2 mM) or quercetin (25 µM) following a 2 hour reaction. The full-length substrate and cleavage product are indicated. C. Autoradiograph of IRE1 (aa 658–1115) (0.5 µM) incubated with [32P]γ-ATP for the indicated time in the presence of ADP (30 µM) or quercetin (30 µM). A Coomassie stain of the same gel is shown below. D. Autoradiograph depicting the time-dependent cleavage of a 32P-labeled IRE1 substrate (20 nM; as in Figure 2B) by IRE1 (0.5 µM) in the presence of ADP (2 mM), quercetin (25 µM) or both. E. Plot of the initial rate of IRE1 RNase activity as a function of quercetin concentration in the absence (filled circles) and presence (open squares) of ADP (2 mM). (see also Supplemental Figure 1).
Figure 3
Figure 3. The crystal structure of IRE1–bound by quercetin and ADP reveals a second ligand binding site, the Q-site
A. Structure of IRE1 (658–1115, Δ869–892) crystallized in the presence of both ADP and quercetin shown in ribbon format (PDB: 3LI0). The kinase domain N- and C-terminal lobes of each protomer in the dimer are green and orange, respectively, and the KEN domains are blue. The kinase and the KEN domain dimer interfaces are indicated. ADP and quercetin are shown in a ball-and-stick representation. B. Electron density (blue-wire mesh) and tube representation of regions of the IRE1:ADP:quercetin ternary complex that are unstructured in the IRE1:ADP dimer (PDB: 2RIO) are shown in stereo view (two left panels) and superimposed on the structure of the same segments in PBD: 3FBV. Left – Amino acids 837–844 including phosphorylated residues S840, S841, and T844 in the activation segment of IRE1 are shown with side chains displayed in a ball and stick format. Right – Amino acids 1036–1042 from the α3’ helix of the KEN domain are shown with side chain residues in ball and stick format. Both structured regions show significant overlap with the previous crystal structure of oligomeric IRE1 (PDB – 3FBV). C. Stereo view of the two-fold symmetric quercetin binding pocket (Q-site). The solvent accessible surface of the KEN domain is shown in light blue and dark blue for the two protomers. Quercetin is shown in a ball-and-stick representation. D. Structure of the Q-site with the residues from the two protomers (purple and green) that interact with quercetin (shown as ball-and-stick in the same view as Figure 3E). Unbiased sigma A-weighted Fo-Fc electron density (see Supplementary Figure 3 for details) for each quercetin molecule is shown as orange or gray wire-mesh. E. A comparison of the Q-site from IRE1:ADP:quercetin (PDB: 3LI0, blue) and IRE1:ADP (PDB: 2RIO, green) shown in a stereo view and depicted as tubes. The residues that line this pocket are shown in ball-and-stick format (the quercetin ligand has been removed from the IRE1:ADP:quercetin structure). F. Schematic depicting the spatial arrangement and interactions of IRE1 and quercetin that define quercetin binding to the Q-site. (also see Supplemental Table 1 and Supplemental Figure 2.)
Figure 4
Figure 4. Mutation of residues lining the Q-site interfere with quercetin-mediated activation of IRE1 RNase
A. Coomassie stained SDS-PAGE of wildtype and mutant yeast IRE1 (aa 658–1115) variants before and after incubation with lambda phosphatase. B. Timecourse of IRE1 RNase activity of IRE1WT (black), IRE1S984E (blue), IRE1K985A (red), and IRE1K992L (green) incubated alone, with ADP (+A; 2 mM), quercetin (+Q; 25 µM) or both (+A +Q). The concentration of each enzyme was adjusted to approximate the activity of IRE1WT in the presence of ADP. C. Bar graph comparing IRE1 RNase activity of IRE1WT, IRE1S984E, IRE1K985A, IRE1K992L, and IRE1F1112L measured as in Figure 4B. The activity of the different enzymes, defined by the fluorescent signal following 1 h treatment, in the presence of ADP was normalized to 1. D. Domain structure of the human-yeast IRE1 chimera (hyIRE1). The rabbit serum used to detect the endogenous and chimeric proteins recognizes the juxta-membrane region as indicated. E. RT-PCR analysis of XBP1 mRNA purified from wild-type MEFs (+/+) or IRE1 knockout cells transduced with empty vector (Mock) or hyIRE1WT exposed to tunicamycin (Tm; 2.5 µg/mL; 4h), DTT (2 mM; 1 h) or thapsigargin (Tg; 0.5 µM; 1h). The position of the unspliced (XBP1U) and spliced (XBP1S) product is indicated. F. RT-PCR analysis of XBP1 mRNA purified from wild-type MEFs (+/+) or IRE1 knockout cells transduced with empty vector (Mock), hyIRE1WT, hyIRE1S984E, or hyIRE1K985A and exposed to 450 µM quercetin for 2 h or thapsigargin (Tg; 0.5 µM for 2h), as indicated. G. Immunoblot of endogenous IRE1 or the hyIRE1 chimeras immunoprecipitated from lysates of the cells shown in Figure 4F. The hyIRE1 chimera migrates slower than endogenous IRE1 due to the larger size of the yeast kinase and endonuclease domains. The anti-eIF2α immunoblot from the flow-through of the immunoprecipitation reaction (lower panel) serves as a loading control. (also see Supplemental Figure 3).
Figure 5
Figure 5. The hierarchy of IRE1 activation by flavonols supports a model of ligand binding in the syn orientation of the prime ring
A. Chemical structures of flavonol-based analogs of quercetin. Quercetin and other relevant flavonols are shown with the 3- and 3’-moieties in the syn orientation; except morin, which is shown in the anti conformation due to clash between the 3- and 2’-hydroxyls. B. Timecourse of RNase activity of IRE1 (0.5 µM) incubated in the presence of various flavonols (25 µM). The activity of IRE1 in the absence of flavonol is indicated by the x symbols. C. Timecourse of RNase activity of IRE1 (0.5 µM) incubated in the presence of both ADP (2 mM) and flavonols (25 µM). The activity of IRE1 in the absence of flavonol is indicated by the x symbols. D. Autoradiograph of IRE1 (0.5 µM) incubated with [32P]γ-ATP in the presence of the known kinase inhibitor staurosporine (25 µM, a positive control for inhibition) or the flavonols (25 µM), following either 10 or 20 min reaction, as indicated. A Coomassie stain of the 20 min gel is shown below. The asterisk indicates the presence of an activity in the sample containing myrecetin that reproducibly degrades IRE1. E. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IRE1 knockout cells transduced with hyIRE1WT following exposure to thapsigargin (Tg; 0.5 µM for 2 h) or flavonols (450 µM for 2 h), as indicated. F. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IRE1 knockout cells transduced with hyIRE1WT, hyIRE1S984, hyIRE1K985A or empty vector (Mock) following exposure to thapsigargin (Tg, 0.5 µM for 2h) or Luteolin (450 µM for 2 h), as indicated. (also see Supplemental Movie 1).
Figure 6
Figure 6. Quercetin increases the population of IRE1 dimers
A. Coomassie stained SDS-PAGE of yeast IRE1 (aa 658–1115, 5 µM) incubated with ADP (2 mM), quercetin (25 µM) or both in the absence (−DSS) or presence of 200 µM disuccinimidyl suberate (+DSS), an irreversible chemical cross-linker. B. Coomassie stained SDS-PAGE of IRE1K985A and IRE1WT incubated with ADP (2 mM), quercetin (25 µM) or both in the presence of 200 µM DSS. C. Comassie stained SDS-PAGE of IRE1 (5 µM) incubated with ADP (2 mM) and the flavonols (25 µM) in the presence of DSS (200 µM). D. Sedimentation distribution of yeast IRE1 (aa 658–1115, 5 µM; black) in the presence of ADP (2 mM; red), quercetin (25 µM; blue) or both (green), as determined by sedimentation velocity analytical ultracentrifugation. Approximate molecular weights are indicated. E. Coomassie stained SDS-PAGE of IRE1WT and IRE1D723A incubated without or with lambda phosphatase. F. Fluorescence timecourse of RNase activity of IRE1WT (black) and IRE1D723A (red) in the presence of ADP (2 mM), quercetin (Q; 25 µM) or both. G. Coomassie stained SDS-PAGE of IRE1WT and IRE1D723A (5 µM) incubated with ADP (2 mM), quercetin (25 µM) or both in the presence of 200 µM DSS.
Figure 7
Figure 7. The nucleotide binding site and the quercetin binding pocket interact to regulate IRE1 RNase
A. Timecourse of IRE1 RNase activity incubated without or with lambda phosphatase (λpp) in the presence of ADP (2 mM) and quercetin (Q; 25 µM). B. Comassie-stained SDS-PAGE of IRE1WT and IRE1D797A incubated with or without lambda phosphatase. C. Fluorescence timecourse of RNase activity of IRE1WT (filled symbols) and IRE1D797A (open symbols) incubated with ADP (2 mM), quercetin (Q; 25 µM) or both. D. Quantification of RNase activity for IRE1D797A incubated with quercetin (25 µM) and increasing concentrations of ADP. The initial rate of IRE1 RNase activity is plotted against ADP concentration (the initial rate of IRE1D797A incubated with quercetin (25 µM) alone is shown by the dashed line). (also see Supplemental Figure 7).
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