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1011.Full.Pdf Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/10/05/mol.115.100917.DC1 1521-0111/88/6/1011–1023$25.00 http://dx.doi.org/10.1124/mol.115.100917 MOLECULAR PHARMACOLOGY Mol Pharmacol 88:1011–1023, December 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1a Endoribonuclease Activity s Nestor O. Concha, Angela Smallwood, William Bonnette, Rachel Totoritis, Guofeng Zhang, Kelly Federowicz, Jingsong Yang, Hongwei Qi, Stephanie Chen, Nino Campobasso, Anthony E. Choudhry, Leanna E. Shuster, Karen A. Evans, Jeff Ralph, Sharon Sweitzer, Dirk A. Heerding, Carolyn A. Buser, Dai-Shi Su, and M. Phillip DeYoung Oncology R&D (K.F., J.Y., L.E.S., K.A.E., J.R., D.A.H., C.A.B., D.S.S, M.P.D.), Biological Sciences (R.T., G.Z., H.Q., S.C., A.E.C., S.S.), and Chemical Sciences, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania (N.O.C., A.S., W.B., N.C.) Downloaded from Received July 20, 2015; accepted September 25, 2015 ABSTRACT Activation of the inositol-requiring enzyme-1 alpha (IRE1a) pro- kinase activation loop to the DFG-out conformation. Inactiva- tein caused by endoplasmic reticulum stress results in the tion of IRE1a RNase activity appears to be caused by a molpharm.aspetjournals.org homodimerization of the N-terminal endoplasmic reticulum conformational change, whereby the aC helix is displaced, luminal domains, autophosphorylation of the cytoplasmic ki- resulting in the rearrangement of the kinase domain-dimer nase domains, and conformational changes to the cytoplasmic interface and a rotation of the RNase domains away from each endoribonuclease (RNase) domains, which render them func- other. In contrast, staurosporine binds at the ATP-binding site tional and can lead to the splicing of X-box binding protein 1 of IRE1a, resulting in a dimer consistent with RNase active (XBP 1) mRNA. Herein, we report the first crystal structures of the yeast Ire1 dimers. Activation of IRE1a RNase activity appears cytoplasmic portion of a human phosphorylated IRE1a dimer to be promoted by a network of hydrogen bond interactions in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)- between highly conserved residues across the RNase dimer 2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1a- interface that place key catalytic residues poised for reaction. selective kinase inhibitor, and staurosporine, a broad spectrum These data implicate that the intermolecular interactions be- at ASPET Journals on September 25, 2021 kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)- tween conserved residues in the RNase domain are required for 2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the ki- activity, and that the disruption of these interactions can be nase and RNase activities of IRE1a. The inhibitor interacts with achieved pharmacologically by small molecule kinase domain the catalytic residues Lys599 and Glu612 and displaces the inhibitors. Introduction (Harding, et al., 2002; Ron, 2002; Feldman et al., 2005). UPR signaling also regulates cell survival by modulating apoptosis and Cellular stresses, such as accumulation of unfolded pro- autophagy and can induce cell death under prolonged ER stress if teins, hypoxia, glucose deprivation, depletion of endoplasmic the misfolded protein burden is too high (Ma and Hendershot, reticulum (ER) calcium levels, and changes in ER redox status 2004; Rouschop et al., 2010; Woehlbier and Hetz, 2011). activate the unfolded protein response (UPR), an intracellular Three key ER membrane proteins have been identified as signal transduction network involved in restoring protein primary effectors of the UPR: protein kinase R–like ER kinase homeostasis [reviewed by Walter and Ron (2011)]. To allevi- (PERK), inositol-requiring enzyme-1 (IRE1) a/b,andactivating ate these types of stress responses, the UPR responds by transcription factor 6 (Schroder and Kaufman, 2005). IRE1a is a halting protein translation, activating transcription of UPR- transmembrane protein that functions both as an ER stress associated target genes, and degrading misfolded proteins sensing receptor via its N-terminal ER luminal domain and as a signal transducer via its cytoplasmic C-terminal kinase and All authors are past or present employees of GlaxoSmithKline. No potential endoribonuclease (RNase) domains (Tirasophon et al., 1998). conflicts of interest were disclosed by the authors. Upon sensing ER stress, the extracellular portion of the IRE1a dx.doi.org/10.1124/mol.115.100917. s This article has supplemental material available at molpharm. protein will homodimerize, allowing for transautophosphoryla- aspetjournals.org. tion, which, in turn, induces a conformational change, resulting ABBREVIATIONS: APY29, 2-N-(3H-benzimidazol-5-yl)-4-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine; BHQ-1, Black Hole quencher-1; ER, endoplasmic reticulum; FAM, 6-carboxyfluorescein fluorescent reporter; FBS, fetal bovine serum; GSK2850163, (R)-2-(3,4-dichlorobenzyl)-N-(4- methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide; ID, identity; IRE1, inositol-requiring enzyme-1; KIRA6, 1-(4-[8-amino-3-tert-butylimidazo(1,5-a) pyrazin-1-yl]naphthalen-1-yl)-3-[3-(trifluoromethyl)phenyl]urea; PCR, polymerase chain reaction; PDB, Protein Data Bank; PERK, protein kinase R–like endoplasmic reticulum kinase; pIRE1a, phosphorylated inositol-requiring enzyme-1 alpha; RNase, endoribonuclease; RT, real time; SAR, structure activity relationship; STS, staurosporine; TEV, tobacco etch virus; UPR, unfolded protein response; UPRE, unfolded protein response element; XBP 1, X-box binding protein 1. 1011 1012 Concha et al. in activation of the RNase domains (Ali et al., 2011). Phosphor- that this process is conserved evolutionarily. Here, we present ylation within the kinase activation loop is an essential step for a proposed structure of the final state of a human phosphor- RNase activation (Prischi et al., 2014). Mammalian IRE1a ylated (active) IRE1a dimer that is cocrystallized with two excises a 26–base pair intron from the mRNA of X-box binding kinase inhibitors that have opposing effects to the RNase protein 1 (XBP 1), which causes a translational frame shift activity of the protein. downstreamofthesplicesitetoproduceXBP1s,theactiveform of the transcription factor (Yoshida et al., 2001; Calfon et al., 2002; Lee et al., 2002). XBP 1 is responsible for the activation of Materials and Methods key UPR target genes, including molecular chaperones and IRE1a Protein Expression and Purification. The cytosolic components of the ER-associated protein degradation machin- domain of human IRE1a (NM_001433), encompassing amino acids ery (Lee at al., 2003). Activation of IRE1a is also reported to 547–977, was cloned into pENTR/tobacco etch virus (TEV)/D-TOPO result in the induction of regulated IRE1a-dependent degrada- (Life Technologies, Carlsbad, CA) and subsequently transferred to a tion of a subset of mRNAs encoding secretory proteins or the pDest8 (Life Technologies) vector backbone containing the N-terminal induction of apoptosis via IRE1a signaling through its kinase Flag epitope tag followed by the 6xHis tag and TEV cleavage site domain and downstream effectors ASK1, JNK1, and Caspase-12 (ENLYFQG/S). Baculovirus generation was accomplished using (Urano et al., 2000; Hollien and Weissman, 2006). the Bac-to-Bac baculovirus generation system (Life Technologies). Flag-His -TEV-IRE1a (547–977) protein expression in baculovirus- Downloaded from Loss of ER homeostasis (i.e., loss or hyperactivation of 6 infected insect cells was accomplished following established procedures UPR signaling) has been attributed to a number of dis- (Wasilko and Lee, 2006). Briefly, a proprietary Sf9 insect cell line eases, including cancer, diabetes, cardiovascular diseases, was grown to the early log phase, infected with 1 Â 107 baculovirus- liver diseases, and neurodegenerative disorders, and the UPR infected insect cells/10 l culture, and incubated at 27°C. Cell paste was is increasingly becoming an attractive pathway in drug harvested at 66–72 hours postinfection. A human phosphorylated or discovery (Hetz et al., 2013; Maly and Papa, 2014). To this dephosphorylated IRE1a protein containing N-terminal Flag-His6 end, an increasing body of work has been performed to identify tags and a TEV protease cleavage site between the tags and an IRE1a molpharm.aspetjournals.org potent and selective molecules of IRE1a and better under- protein was purified from ∼150 g of cells from a 10-l culture [lysed in stand how these molecules bind and affect IRE1a activa- 1.5 l of lysis buffer (50 mM Hepes, pH 7.5, 10% glycerol, and 300 mM tion mechanisms. Crystal structures of the C-terminal region NaCl) by the EmulsiFlex-C50 homogenizer (Avestin, Ottowa, ON, Canada)]. The protein in the clear supernatant from centrifugation at of phosphorylated (active) yeast Ire1 were the first to be char- 30,000g for 30 minutes at 4°C was first captured in 20 ml of NiNTA-SF acterized and revealed that Ire1 forms dimers arranged in beads (Qiagen, Venlo, Netherlands) in batch mode for 4 hours at 4°C. “ ” a back-to-back configuration, with the kinase active sites The beads were poured into a column and washed with 20 mM facing outward [Protein Data Bank (PDB) identity (ID) 2RIO; imidazole in lysis buffer, and the IRE1a protein was eluted from the Lee at al., 2008; PDB ID 3FBV; Korennykh et al., 2009; PDB column by 300 mM imidazole
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