ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B

•Mutually antagonistic binding of phosphorylated eIF2 and ISRIB to eIF2B•Opposing structural rearrangement eIF2B by or eIF2•Phosphorylated exposes ISRIB’s ability enhance activity in vitro•Weak ISR-inhibitory cells lacking The small molecule antagonizes the activation integrated stress response (ISR) translation initiation factor 2, eIF2(?P). eIF2(?P) bind distinct sites their common target, eIF2B, a guanine nucleotide exchange for eIF2. We have found that ISRIB-mediated acceleration eIF2B’s vitro is observed preferentially presence attenuated mutations desensitize inhibitory effect efficacy as an ISR inhibitor also depends on Cryoelectron microscopy (cryo-EM) showed engagement both regulatory two molecules remodels ISRIB-binding pocket pockets would engage eIF2? during active exchange, thereby discouraging events. In vitro, reciprocally opposed each other’s eIF2B. These findings point allostery action culminating inhibition ISR. Under diverse stressful conditions, ?-subunit eukaryotic 2 (eIF2) serine 51 its N-terminal domain (NTD). This converts eIF2, substrate eIF2B; (GEF) reactivates heterotrimer accelerating release GDP from ?-subunit with GTP (Ranu London, 1979Ranu R.S. London I.M. Regulation protein synthesis rabbit reticulocyte lysates: additional required formation ternary complex (eIF-2.GTP.Met-tRNAf) demonstration heme-regulated kinase.Proc. Natl. Acad. Sci. USA. 1979; 76: 1079-1083Crossref PubMed Scopus (56) Google Scholar; de Haro et al., 1996de C. Méndez R. Santoyo J. eIF-2alpha kinases control synthesis.FASEB 1996; 10: 1378-1387Crossref (231) Scholar), thus promoting initiator methionyl-tRNA (Met-tRNAi) eIF2?GTP (Dev 2010Dev K. Qiu H. Dong Zhang F. Barthlme D. Hinnebusch A.G. beta/Gcd7 subunit 2B (eIF2B), factor, crucial vivo.Mol. Cell. Biol. 2010; 30: 5218-5233Crossref (27) Scholar). By depleting complexes GTP, Met-tRNAi cell, phosphorylation attenuates most mRNAs, important effects synthesis. However, few mRNAs increased phosphorylation-dependent manner. As latter encode potent transcription factors, production [eIF2(?P)] coupled conserved gene expression program referred (Harding 2003Harding H.P. Y. Zeng Novoa I. Lu P.D. Calfon M. Sadri N. Yun Popko B. Paules al.An regulates amino acid metabolism resistance oxidative stress.Mol. 2003; 11: 619-633Abstract Full Text PDF (2205) homeostatic pathway contributes organismal fitness (Pakos-Zebrucka 2016Pakos-Zebrucka Koryga Mnich Ljujic Samali A. Gorman A.M. response.EMBO Rep. 2016; 17: 1374-1395Crossref (821) some circumstances, heightened associated unfavorable outcomes, motivating search inhibitors. When applied administered animals, drug-like molecule, ISRIB, disrupts (Sidrauski 2013Sidrauski Acosta-Alvear Khoutorsky Vedantham P. Hearn B.R. Li Gamache Gallagher C.M. Ang K.K. Wilson al.Pharmacological brake-release mRNA enhances cognitive memory.eLife. 2013; 2: e00498Crossref (357) Scholar) has been reported exert beneficial models neurodegeneration (Halliday 2015Halliday Radford Sekine Moreno Verity le Quesne Ortori C.A. Barrett D.A. Fromont Fischer P.M. al.Partial restoration rates prevents without pancreatic toxicity.Cell Death Dis. 2015; 6: e1672Crossref (174) Zhu 2019Zhu P.J. Khatiwada S. Cui Reineke L.C. Dooling S.W. Kim J.J. W. Walter Costa-Mattioli Activation mediates behavioral neurophysiological abnormalities Down syndrome.Science. 2019; 366: 843-849Crossref (50) head injury (Chou 2017Chou Krukowski Jopson T. Rosi Inhibition reverses deficits after traumatic brain injury.Proc. 2017; 114: E6420-E6426Crossref (91) dysmyelination (Wong 2018Wong Y.L. LeBon L. Edalji Lim H.B. Sun Sidrauski rescues stability Vanishing White Matter Disease mutant complexes.eLife. 2018; 7: e32733Crossref (54) Abbink 2019Abbink T.E.M. Wisse L.E. Jaku E. Thiecke M.J. Voltolini-González Fritsen Bobeldijk Ter Braak T.J. Polder Postma N.L. al.Vanishing white matter: deregulated therapy target.Ann. Clin. Transl. Neurol. 1407-1422Crossref (24) does not affect levels eIF2(?P), indicating site downstream this effector. ISRIB-resistant were mapped genetically ?- ?-subunits disrupt high-affinity (Kd ~10 nM) surface (Sekine 2015Sekine Zyryanova Crespillo-Casado Harding Ron Stress responses. Mutations identify target memory-enhancing compound.Science. 348: 1027-1030Crossref (143) Tsai 2018Tsai J.C. Miller-Vedam Anand A.A. Jaishankar Nguyen H.C. Renslo A.R. Frost Structure reveals mechanism molecule.Science. 359: eaaq0939Crossref (82) 2018Zyryanova A.F. Weis Faille Alard Allen Parts al.Binding eIF2B.Science. 1533-1536Crossref (78) demonstrating target. ~500 kDa decamer, assembled sets five subunits (Kashiwagi 2016Kashiwagi Takahashi Nishimoto Hiyama T.B. Higo Umehara Sakamoto Ito Yokoyama Crystal structure 2B.Nature. 531: 122-125Crossref (64) It catalytic sites, comprised bipartite ?-subunit whose domains embrace nucleotide-binding eIF2? enforcing conformation favors dissociation GTP. Engagement unphosphorylated catalytically productive NTD (eIF2?-NTD) between ~100 Å 2019Kashiwagi Yonemochi Shirouzu Structural basis response.Science. 364: 495-499Crossref (38) Kenner 2019Kenner L.R. Myasnikov Klose C.J. McGeever L.A. eIF2B-catalyzed phosphoregulation 491-495Crossref phosphorylated, eIF2?-NTD (P-eIF2?-NTD) engages at alternative site, ?- (Adomavicius 2019Adomavicius Guaita Zhou Jennings M.D. Latif Z. Roseman Pavitt G.D. translational phosphorylation.Nat. Commun. 2136Crossref (52) Gordiyenko 2019Gordiyenko Llácer J.L. Ramakrishnan V. through 2640Crossref Kashiwagi Scholar): nonproductive mode inhibits binds single center symmetry, interface (Tsai (see Figure 7A, below). stands reason Indeed, when added crude preparations accelerates 2015Sidrauski Kampmann Sokabe Mendez A.S. Newton B.W. Tang E.L. dimerization response.eLife. 4: e07314Crossref (159) A simple proposed account such stimulation: decameric consists one ?2 dimer ???? tetramers; across tetramers, decamer assembly stability. According model, which well supported features increasing effective concentration active, Accelerated be favored large pool unassembled cell. Yet, fractionation mammalian cell lysates density gradient centrifugation suggested existence pools precursor Being slow method, might fail detect precursors migrating predicted position gradient, if rapid equilibrium decamers. finding little no pure decamers speaks against enzyme stabilizing equilibrium. considerations, hints differences eIF2?-binding (Gordiyenko prompted us examine evidence modes action. Here, we report biochemical, structural, cell-based allosterically inhibit To assess isolation loaded BODIPY-GDP onto eIF2(?S51A) isolated 293-F expressing non-phosphorylatable S51A mutation ?-subunit. then utilized fluorescence-based assay recombinant human previously only minimally accelerated mediated devoid (Figure 1A). Introduction into directed toward eIF2(?S51A)?BODIPY-GDP. was significantly, although partially, reversed 1B), eIF2B? residues, E310K L314Q (Kimball 1998Kimball S.R. Fabian J.R. Jefferson L.S. eIF2alpha. Role alpha- delta-subunits eiF2b.J. Chem. 1998; 273: 12841-12845Abstract (79) introduced genome cultured CHO (by CRISPR/Cas9-mediated homologous recombination), corresponding hamster residues (eIF2B? E312 L316) imparted ISR-insensitive phenotype, reflected blunted stress-induced ISR-responsive CHOP::GFP reporter 1C) repression synthesis, normally stressed 1D). are consistent phenotype yeast, GCD2E377K GCD2L381Q (Pavitt 1997Pavitt Yang Homologous segments three mediate regulation eIF2.Mol. 1997; 1298-1313Crossref (104) equivalent substitutions eIF2B?, wild-type 1B). Together, these observations indicate that, relevant vivo. recently published structures eIF2?eIF2B suggest alters resolution limits confidence conclusion, especially regard site. Therefore, collected further cryo-electron images trimer solved new variant structures: resolved (the ?P1 complex), trimers ends (?P2 ?-subunits end other ?P? complex) (Table 1; S1). overall similar those subunits’ almost identical previous but improved resolution. addition, re-analyzed dataset eIF2B?eIF2(?P) extracted apo particles S1).Table 1Cryo-EM Data Collection Image Processing?P1 (EMDB-30570) (PDB 7D45)?P2 (EMDB-30569) 7D44)?P? (EMDB-30568) 7D43)eIF2B (EMDB-30571) 7D46)Data collection processingMicroscopeTecnai ArcticaTecnai ArcticaCameraK2 SummitK2 SummitMagnification23,50023,500Voltage (kV)200200Electron exposure (e–/Å2)5050Exposure per frame1.251.25Number frames collected4040Defocus range (?m)?1.5 ?3.1?1.5 ?3.1Micrographs7,7294,987Pixel size (Å)1.471.473D Processing packageRELION-3.0RELION-2.1,3.0Symmetry imposedC1C1Initial particle images1,889,1011,482,123Final images208,72880,92166,721330,601Initial reference map5B04 (40 Å)5B04 Å)Map resolutionMasked (FSC = 0.143)3.774.014.283.97Map sharpening B-factor?119.0?103.6?108.8?165.6RefinementInitial model used6O9Z, 6K726O9Z, 6K72Model compositionNon-hydrogen atoms27,52928,39031,00526,636Protein residues3,6593,8254,3473,481B factorsProtein26.7437.4771.8323.55RMSDBond lengths (Å)0.0090.0060.0060.008Bond angles (°)0.9160.8500.9750.928ValidationMolProbity score2.482.482.642.47Clashscore19.5320.1627.6418.44Poor rotamers (%)0.350.280.310.42CaBLAM outliers (%)5.785.196.796.08Ramachandran plotFavored (%)83.1383.6881.6582.43Allowed (%)16.7616.2618.3017.52Disallowed (%)0.110.050.050.06Map CC (CCmask)0.800.780.790.82See S1. Open table tab See compared, en bloc tetrameric unit 2A). widens gap eIF2B? otherwise accommodate (Figures S2A) consequence, C? atoms helix ?3 (helix ?-?3, 247–267), intensively interact eIF2B?-side gap, displaced 3.2 (on average) helical axis rotated 7.9° away root-mean-square deviation [RMSD] alignment 0.7 Å) 2B, right panel). widening ?P2 (average displacement ?-?3 2.8 rotation 8.1°) S2A S2B). arrangement more accommodates (PDB: 6O81) (Kenner minor narrowing following accommodation average 1.1 2.3° eIF2B?) S2A). suggests upon Such appears feature, because gaps yeast bound wider than Widening complexed (P-eIF2?) 6O9Z) conspicuous presently determined 2.4 6O9Z versus seems make contribution Contacts eIF2B? may contribute difference, significance needs exploration 2A induced affects ISRIB. formed heterodimeric units Comparing eIF2B?ISRIB 6CAJ) relative altered. around (within 10 ?? (eIF2B? 2C) 2.2 ???? (eIF2B?? eIF2B?? complex. includes key involved 2C S2C)