The structural basis of translational control by eIF2 phosphorylation

Abstract

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.

Fig. 1

Electron cryomicroscopy (cryoEM) structure of the eIF2B/eIF2αP complex. Left: Refined 3.9 Å cryoEM map of eIF2B/eIF2αP complex with central eIF2B decamer and two lateral eIF2 trimers. Local amplitude scaling (Locscale) was used to apply local density re-scaling based on a fitted model. Right: modelled densities displayed as secondary structures. a Top and b back views shown. Map surface and subunits are coloured as indicated, with the regulatory phosphoserine in red. Scale bar relates to cryoEM map and is 20 Å. The figure was drawn with UCSF Chimera software

Fig. 2

eIF2α conformational flexibility on binding to eIF2B. a eIF2α conformation differs between eIF2αP/eIF2B and ternary complexes (TCs) complexes. eIF2α from our eIF2αP/eIF2B complex (domains 1 and 2 shown in gold and arrowed, domain 3 grey) aligned onto TC from 3JAP (2α domains 1–3 in grey) using eIF2α domain 3 as a reference. b–g Flexibility between eIF2α domains 2 and 3 seen in eIF2αP/eIF2B 3D classes obtained when halves of the particles were independently classified using a localised reconstruction script (described in the Methods section and Supplementary Fig. 2b). h eIF2αP conformations from b–g aligned modelled onto 3JAP eIF2 (as in a) as semi-transparent ribbons. Dashed black arrows indicate changed positions. In a, h, Ser52 and Ser52(P) side chains are shown in red

Fig. 3

eIF2α N-terminal domain binds between the eIF2B α and δ subunits. a Overview and b detail of eIF2αP molecular interactions with eIF2B, showing the electron cryomicroscopy (cryoEM) density as a mesh and highlighting a network of arginines around Ser52 and the KGYID sequence. c, d Complimentary surface potentials at the eIF2αP/eIF2B interface. c Electrostatic surface representation of eIF2Bαβδ and d Coulombic potential due to eIF2αP displayed at the eIF2Bαβδ surface. Blue, positive and red, negative

Fig. 4

eIF2B antagonises RNA -activated protein kinase (PKR) activity. a Western blot of eIF2α phosphorylation (1 µM eIF2) by PKR (10 nM), when incubated in the presence of increasing concentrations of eIF2B. eIF2α and eIF2αP resolved by Phos-tag acrylamide gel electrophoresis. Both panels are from the same blot. Experiment repeated four times with similar results. Source data are provided in the Source Data file. b Quantification of eIF2αP percentage

Fig. 5

Non-phosphorylated eIF2/eIF2B complex is almost identical to eIF2αP/eIF2B. a Overview of eIF2/eIF2B structure map (after local amplitude scaling (Locscale)). Orientation and surface coloured as in Fig. 1a. Scale bar is 20 Å. b Model fitting to electron cryomicroscopy (cyoEM) maps around Ser52 in eIF2/eIF2B (left) and eIF2αP/eIF2B (right) complexes, indicating some minor differences between complexes

Fig. 6

Model for eIF2B GDP exchange and ternary complex (TC) formation. A series of steps for guanine nucleotide exchange factor (GEF) action and recruitment of initiator tRNA to form TC based on our structures and docking of the GEF domain (PDB 1PAQ), GDP (PDB 4RD6) or initiator tRNA (3JAP, chain 1), as outlined in the main text and Supplementary Movie 2. Grey arrows indicate steps affected by eIF2(αP)