Novel mechanisms of eIF2B action and regulation by eIF2α phosphorylation

Abstract

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric GTPase, which plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP•Met-tRNAi ternary complex (TC), which is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, which triggers the integrated stress response (ISR). Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms of eIF2B action and regulation remain unknown. Here we employ a combination of NMR, fluorescence spectroscopy, site-directed mutagenesis, and thermodynamics to elucidate the mechanisms of eIF2B action and its regulation by phosphorylation of the substrate eIF2. We present: (i) a novel mechanism for the inhibition of eIF2B activity, whereby eIF2α phosphorylation destabilizes an autoregulatory intramolecular interaction within eIF2α; and (ii) the first structural model for the complex of eIF2B with its substrate, eIF2-GDP, reaction intermediates, apo-eIF2 and eIF2-GTP, and product, TC, with direct implications for the eIF2B catalytic mechanism.

Figure 1.

Intramolecular interaction in eIF2α. (A) TROSY-HSQC spectra of ²H/¹⁵N-labeled eIF2α (black), eIF2α-NTD (blue), and ²H/¹⁵N/¹³C-labeled eIF2α-CTD (red). (B) Boxed area in (A). The arrows indicate movement of peaks away from their full length WT positions. (C) Plot of chemical shift perturbations (CSP) between eIF2α and its individual domains. Gray bars represent indeterminate residues for which no analysis could be performed. Key structural features are shown above the plot for reference, with the extreme end (aa 302–314) of the C-terminal tail (CTT) shown as a wavy line. (D) Residues affected by the intramolecular interaction mapped onto the structure of eIF2α, colored from yellow (>1 standard deviation (σ)) to red (>3σ). Indeterminate residues are colored gray. Residues <1σ are colored black. Residues 302–314 were absent in the NMR structure and are thus not displayed here. (E) Model for the closed conformation of eIF2α shown in ribbon and colored as in panel D.

Figure 2.

Effects of the phosphomimetic S51D mutation in eIF2α. (A) TROSY-HSQC spectra of ²H/¹⁵N-labeled eIF2α (black) and a phosphomimetic mutant, eIF2α_S51D (purple). (B) The boxed area in (A). The arrows indicate movement of peaks away from their WT positions. (C) Plot of chemical shift perturbations between eIF2α and eIF2α_S51D. Gray bars represent indeterminate residues for which no analysis could be performed. (D) Residues affected by the phosphomimetic mutation mapped onto the structure of eIF2α, colored from yellow (>1σ) to red (>3σ). Indeterminate residues are colored gray. Residues <1σ are colored black. Comparison to Figure 1D reveals that the effects of the phosphomimetic mutation overlap substantially with the intramolecular surface. (E) The same boxed area as in (B), but with the addition of a third overlain spectrum of ²H/¹⁵N/¹³C-labeled eIF2α-CTD (red). Peaks corresponding to residues affected by the phosphomimetic mutation displayed a consistent pattern of movement, where they are located halfway between the peak positions in the eIF2α spectrum (‘bound’ state) and the eIF2α-CTD spectrum (‘free’ state). Arrows indicate movement of peaks away from their full length WT positions. (F) Residues affected by the phosphomimetic mutation mapped onto the structure of eIF2α-NTD. Coloring scheme is the same as in (D).

Figure 3.

eIF2α-NTD surfaces affected by binding to eIF2Bα. (A) TROSY-HSQC spectra of ²H/¹⁵N-labeled eIF2α-NTD, free (black) and in the presence of excess unlabeled eIF2Bα (red). (B) The boxed area in (A). Some of the peaks experiencing selective signal loss are labeled. (C) Plot of signal loss for residues in eIF2α-NTD. Gray bars represent indeterminate residues for which no analysis could be performed. (D) Residues experiencing selective signal loss mapped onto the structure of eIF2α-NTD, colored from yellow (>1σ) to red (>3σ). Indeterminate residues are colored gray. Residues <1σ are colored black.

Figure 4.

eIF2α-NTD surfaces affected by binding to eIF2Bβ. (A) TROSY-HSQC spectra of ²H/¹⁵N-labeled eIF2α-NTD_S51D, free (black) and in the presence of excess unlabeled eIF2Bβ (red). (B) The boxed area in (A). Some of the peaks experiencing selective signal loss and/or chemical shift perturbations are labeled. (C) Plot of signal loss for residues in eIF2α-NTD_S51D. Gray bars represent indeterminate residues for which no analysis could be performed. (D) Residues experiencing selective signal loss mapped onto the structure of eIF2α-NTD, colored from yellow (>1σ) to red (>2σ). Residues experiencing chemical shift perturbations of >0.01ppm, but without significant selective signal loss (<1σ), are colored in gold. Indeterminate residues are colored grey. Residues <1σ are colored black. Comparison to the intramolecular surface in Figure 1D reveals overlap between the eIF2Bβ binding surface and the intramolecular interface.

Figure 5.

eIF2Bα, eIF2Bβ, and eIF2B_reg bind eIF2α-NTD and eIF2α-NTD_S51D with similar affinities. Plot of the changes in fluorescence anisotropy of fluorescein-labeled eIF2α-NTD (black) and eIF2α-NTD_S51D (purple) in the presence of increasing concentrations of unlabeled eIF2Bα (solid squares) and eIF2B_reg (circles) (A), and eIF2Bβ (B). Calculated K_Ds are shown next to the corresponding titration curves. Zoomed-in eIF2B_reg titration curves are shown in the inset of panel A. No substantive difference in affinity was observed for eIF2Bα, eIF2Bβ, or eIF2B_reg as a function of the phosphorylation state of eIF2α-NTD.

Figure 6.

Structural model for the eIF2B/eIF2 interaction. (A) Model of the eIF2B•eIF2α interaction, where eIF2α-NTD is docked into the eIF2B regulatory subcomplex pocket (6). eIF2B subunits are shown in surface representation; eIF2α is shown as ribbon. eIF2Bα/β/δ residues shown to cross-link to both phosphorylated and unphosphorylated eIF2α are red; residues in eIF2Bβ that cross-link only to unphosphorylated eIF2α are orange (6). eIF2Bγ/ϵ residues shown to cross-link to eIF2γ in eIF2B•apo-eIF2 complexes are navy, except the two eIF2Bϵ residues with lower efficiency of cross-linking to eIF2(α-P)-GDP than to apo-eIF2 (6), which are light blue. The sites of CACH/VWM mutations in eIF2Bγϵ are gray. Residues in eIF2α are colored according to the following scheme: (i) residues in the P-loop are purple, unless colored as detailed below; (ii) residues affected by eIF2Bα binding (see Figure 3) are navy; (iii) residues affected by eIF2Bβ binding (see Figure 4) are cyan and (iv) residues affected by both are blue. Inset: zoomed-in view of the eIF2B_reg pocket, with eIF2α-NTD, -CTD, and P-loop labeled. (B) Model of the eIF2B•TC complex, oriented and aligned using cross-linking data (6), as well as a proposed docking of the eIF2Bϵ-CTD catalytic domain (purple, semi-transparent). eIF2B coloring is as in panel (A). TC is shown as ribbon. The nucleotide is displayed in green. Inset: zoomed-in view of the eIF2γ-ibnding surface of eIF2B_cat, rotated 30 degrees as shown. The two eIF2Bγ/ϵ residues shown to cross-link equally well to both apo-eIF2 and eIF2(α-P)-GDP (6) are navy and marked with black circles. The two eIF2Bϵ residues with lower efficiency of cross-linking to eIF2(α-P)-GDP than to apo-eIF2 (6) are light blue and marked with red circles. (C) Model of the eIF2B complex with nucleotide-bound eIF2 in ‘extended’ conformation, obtained by merging the model of the eIF2B•eIF2α complex shown in (A) and the model of the eIF2B•TC complex shown in (B). eIF2B and eIF2α coloring is as in panel (A). (D) Model of the eIF2B•apo-eIF2 complex, in which eIF2β/γ remain docked as in the eIF2B•TC complex shown in (B), but eIF2α is ‘closed’ as per the intramolecular interaction displayed in Figure 1. eIF2B coloring is as in panel (A). Cartoon representation of the complexes is shown on the bottom right of each panel.