Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo–electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

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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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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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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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