Structural basis for eIF2B inhibition in integrated stress response

AbstractA core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers methionylated initiator tRNA to the ribosome in a GTP-dependent manner. In contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor eIF2B, which leads to a deficiency of active eIF2 and resultant global translation repression. To unveil the mechanism by which the eIF2 phosphorylation status regulates the eIF2B nucleotide exchange activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. Intriguingly, the unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active “productive” and nucleotide exchange-inactive “nonproductive” modes, respectively. The nonproductive-mode phosphorylated eIF2, extending from one of the two eIF2B “central cavities”, not only prevents nucleotide exchange on itself, but also sterically prevents unphosphorylated eIF2 from productively binding on the other central cavity of eIF2B, which explains how phosphorylated eIF2 inhibits eIF2B.One Sentence SummaryA drastic change in the binding mode of eIF2 to eIF2B induces translational control in stress.

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Structural basis of eIF2B-catalyzed GDP exchange and phosphoregulation by the integrated stress response

10.1101/504654 ◽

2018 ◽

Author(s):

Aditya A Anand ◽

Lillian R Kenner ◽

Henry C Nguyen ◽

Alexander G Myasnikov ◽

Carolin J Klose ◽

...

Keyword(s):

Stress Response ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Structural Basis ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor Eif2 ◽

General Translation

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a GTPase, that becomes activated by eIF2B, a large two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from substrate into an inhibitor of eIF2B. We report cryoEM structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's catalytic centers to catalyze guanine nucleotide exchange. Phosphorylation refolds eIF2, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequesters it into a non-productive complex.

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ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

10.1101/2020.09.28.316653 ◽

2020 ◽

Author(s):

Alisa F. Zyryanova ◽

Kazuhiro Kashiwagi ◽

Claudia Rato ◽

Heather P. Harding ◽

Ana Crespillo-Casado ◽

...

Keyword(s):

Stress Response ◽

Binding Pocket ◽

Inhibitory Effect ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor 2

AbstractThe small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

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Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4618 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4618-4631 ◽

Cited By ~ 31

Author(s):

J L Bushman ◽

M Foiani ◽

A M Cigan ◽

C J Paddon ◽

A G Hinnebusch

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Eukaryotic Translation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor 2 ◽

Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

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eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

10.1101/2020.12.28.424567 ◽

2020 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

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eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽

10.7554/elife.65703 ◽

2021 ◽

Vol 10 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

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Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4618-4631.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4618-4631

Author(s):

J L Bushman ◽

M Foiani ◽

A M Cigan ◽

C J Paddon ◽

A G Hinnebusch

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Eukaryotic Translation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor 2 ◽

Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

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Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

Nature Communications ◽

10.1038/s41467-021-26164-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Michael Schoof ◽

Lan Wang ◽

J. Zachery Cogan ◽

Rosalie E. Lawrence ◽

Morgane Boone ◽

...

Keyword(s):

Stress Response ◽

Translation Initiation ◽

Viral Proteins ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Double Stranded Rna ◽

Nucleotide Exchange Factor ◽

Initiation Factor Eif2

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

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Viral Evasion of the Integrated Stress Response Through Antagonistic eIF2-P Mimicry

10.1101/2021.06.07.447473 ◽

2021 ◽

Author(s):

Michael Schoof ◽

Lan Wang ◽

J Zachery Cogan ◽

Rosalie Lawrence ◽

Morgane Boone ◽

...

Keyword(s):

Stress Response ◽

Translation Initiation ◽

Viral Proteins ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Double Stranded Rna ◽

Nucleotide Exchange Factor ◽

Initiation Factor Eif2

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 43 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα; mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.;

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A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

10.1101/2021.12.03.471181 ◽

2021 ◽

Author(s):

Lan Wang ◽

Morgane Boone ◽

Rosalie E Lawrence ◽

Adam Frost ◽

Peter Walter ◽

...

Keyword(s):

Stress Response ◽

Point Mutation ◽

Translational Control ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Nucleotide Exchange Factor

AbstractIn eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.

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