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. 2019 Jun 14;10(1):2640.
doi: 10.1038/s41467-019-10606-1.

Structural basis for the inhibition of translation through eIF2α phosphorylation

Yuliya Gordiyenko  1 , José Luis Llácer  2   3 , V Ramakrishnan  1
Affiliations

Structural basis for the inhibition of translation through eIF2α phosphorylation

Yuliya Gordiyenko et al. Nat Commun. .

Abstract

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Overview of cCryoEM structure of eIF2B–eIF2(αP) complex. a Two views of the overall cryoEM map 2 of eIF2B–eIF2(αP) complex at 4.3 Å resolution with different subunits of the complex colour-coded. b Two views of the cryoEM map A of eIF2B–eIF2(αP) complex at 4.6 Å containing clear density for eIF2β subunit on one side of the complex at the top
Fig. 2
Fig. 2
Contacts of eIF2α with the regulatory eIF2B subunits. a Model of eIF2B–eIF2(αP) complex fitted in maps 1 and 2. b Contacts of eIF2α-D1 with α, β and δ regulatory subunits of eIF2B. Possible residues in contacts with eIF2α Ser51 phosphate (red sticks) are H82, Y304 and R75 in eIF2Bα (shown in yellow sticks). eIF2Bδ E377 (green sticks) in contact with the 56–63 helix (magenta) of eIF2α affected by the phosphate is also shown. eIF2Bδ E377K overcomes the effect of Ser51 phosphorylation and eIF2Bβ I118T and S119P (in brown loop) reduce the effect of phosphorylation. Also shown are residues in S. cerevisiae eIF2B regulatory subunits α, β and δ corresponding to S. pombe residues which cross-linked to eIF2α and residues in eIF2α which cross-linked to eIF2Bα (pink spheres) and to eIF2Bβ (red spheres). c Superposition of eIF2α-D1 in eIF2B–eIF2(αP) complex and in the TC (PDB 3JAP), showing that the same helix 58–63 (coloured magenta) in eIF2α-D1 interacts with both eIF2Bδ and Met-tRNAiMet in a different conformation, suggesting direct competition for eIF2α-D1
Fig. 3
Fig. 3
Contacts of eIF2 β and γ with the catalytic subunits of eIF2B. a eIF2B–eIF2(αP) complex model in spheres representation fitted in map A showing tilted conformation of eIF2γ, which is stabilised by its contact with eIF2Bγ and extended conformation of eIF2β contacting the interface area of the two γ and ε catalytic subunits of eIF2B. b Close-up view of the model fitting into the density of map A. c Modelled positions of eIF2 γ and β subunits after classification, showing extensive movements of these subunits around eIF2B ε and γ PLD domains. For clarity, only two eIF2 models are shown (corresponding to maps A—coloured and C—grey). d Modelled positions of eIF2γ subunit and eIF2α-D3 in all three maps (map 1—orange for eIF2γ and purple for eIF2α-D3, map A—blue, map B—green, map C—grey, map D—yellow). For clarity, only few elements in each of the eIF2γ domains are shown. e Superposition of domain II of eIF2γ in map A (coloured light blue) with that in map 1 (eIF2γ coloured orange) shows the rearrangement of three eIF2γ domains when it is in the tilted conformation. f Conformation of eIF2β fitted in map D (red) is different from the conformations found in three other maps (A - blue, B - green and C - grey). eIF2 γ and α shown are from map D
Fig. 4
Fig. 4
Extra densities in the maps after masked classification in proximity of eIF2γ could accommodate eIF2B ε-cat HEAT domain with eIF2. a Map B of eIF2B–eIF2(αP) complex obtained by masked classification around eIF2 γ and β showing extra density in contact with eIF2γ. b The extra density in map B could accommodate most of the ε-cat HEAT domain and would be in contact with eIF2γ domains III and G away from nucleotide-binding site. ~85 Å distance separates this extra density from the C terminus of the eIF2Bε and is just enough for the 73 residues linker (res. 472–544) to connect ε-cat with the rest of eIF2Bε. c Map D of eIF2B–eIF2(αP) complex obtained by masked classification around eIF2 γ and an extra density seen at a lower threshold (black) in proximity of nucleotide-binding pocket. d The size and shape of the extra density in map D could fully account for the whole ε-cat HEAT domain. Also, in this map, eIF2β approaches the NFD motif (blue spheres) in eIF2Bε
Fig. 5
Fig. 5
Superposition of the TC with eIF2B–eIF2(αP) complex based on eIF2γ. a Superposition of yeast TC (6GSM) in grey with the model of eIF2B–eIF2(αP) complex in map C showing that Met-tRNAiMet can bind without clash to eIF2γ and eIF2α- D3 while eIF2α-D1 is still attached to eIF2B. b Same superposition as in a in a different orientation shows the large conformational changes that eIF2α–D1 and D2 undergo when bound to eIF2B or Met-tRNAiMet both competing for eIF2α. c Same as in b, but superimposed with S. solfataricus TC (3V11, light blue)
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