eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics

Abstract

Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi (Met)) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2β is the most labile of the three subunits (eIF2α/β/γ). We then compared conformational dynamics of the ternary complex eIF2:GTP:Met-tRNAi (Met) with apo eIF2 using comparative chemical cross-linking. Results revealed high conformational dynamics for eIF2α in apo eIF2 while in the ternary complex all three subunits are constrained. Novel post-translational modifications identified here in both eIF2 and eIF2B were combined with established sites, and located within protein sequences and homology models. We found clustering at subunit interfaces and highly phosphorylated unstructured regions, at the N-terminus of eIF2β, and also between the eIF2Bε core and catalytic domains. We propose that modifications of these unstructured regions have a key role in regulating interactions between eIF2 and eIF2B, as well as other eIFs.

Keywords: acetylation; eukaryotic translation initiation; mass spectrometry; phosphorylation.

Figure 1

Mass spectrum of trimeric eukaryotic initiation factor 2 (eIF2). His-tagged eIF2 was purified from yeast. The mass spectrum shows intact trimeric eIF2 (dark blue stars), α/γ and β/γ dimers (light-blue and purple stars), protein subunits (α, green; γ, red; and β, yellow) and stripped α/γ dimer (orange). The dimeric species and γ-subunit show two populations corresponding to bound (filled) and unbound (unfilled) GDP (average Δm 461 Da). The insert shows a tandem mass spectrum of intact eIF2 (charge state 24+). Two stripped complexes were observed, the predominant α/γ dimer (orange) and the low-abundant β/γ dimer (light-green). Highly charged β-subunit (yellow) was also observed.

Figure 2

Homology models of eukaryotic initiation factor 2 (eIF2) protein subunits. Homology models for α- (green), β- (yellow) and γ-subunits (red) are shown. Intra-protein cross-links are shown as dotted lines and cross-linked lysine residues are shown as space fillings (α and β, red; and γ, blue). Cα-distances in Å are given for the projected cross-links.

Figure 3

Cross-linking of eukaryotic initiation factor 2 (eIF2) and formation of the ternary complex. (a) The homology models of α-, β- and γ-subunits were aligned with the archaeal homologue of eIF2 (PDB ID 3CW2). Cross-linking apo eIF2 reveals several interactions between the α-subunit and β- and γ-subunits, showing that eIF2 is highly flexible in solution. (b) Homology models of α-, β- and γ-subunits were aligned with the archaeal homologue of the ternary complex (TC; PDB ID 3V11). Comparative cross-linking of apo eIF2 versus the TC reveals multiple inter-subunit cross-links with reduced intensities after binding Met-tRNA_i^Met. Changes in cross-linking intensities are colour coded; values 2.5–10 represent cross-linking intensities that are higher in eIF2 compared with the TC.

Figure 4

Location of identified post-translational modifications (PTMs) in eukaryotic initiation factor 2 (eIF2). (a) Phosphosites and acetylation sites identified in this or previous studies are shown on the protein subunits of eIF2. The proteins are represented as bars. The arrows (green, yellow and red) represent the parts of the sequence for which homology models could be obtained. (b) Homology models of eIF2-α, -β and -γ aligned with the archaeal homologue (PDB ID 3CW2). Phosphosites (dark blue) and acetylation sites (cyan) are shown as space-filled spheres. Please note that S52 is usually referred to as S51 in literature, which derives from corrected residue numbering after removal of N-terminal methionine. The numbering used here includes N-terminal methionine.

Figure 5

Location of post-translational modifications (PTMs) in eukaryotic initiation factor 2B (eIF2B). Phosphosites and acetylation sites identified in this or previous studies are shown on the protein subunits of eIF2. The proteins are represented as bars. The arrows represent the parts of the sequences for which homology models could be obtained. The magnification shows a phosphosite cluster of eIF2Bε. This cluster is located in a flexible loop connecting the ε/γ dimer (cartoon insert) and the catalytic domain of eIF2Bε.

Figure 6

Eukaryotic initiation factor 2 (eIF2) interactions with initiator tRNA and eIF2B are regulated by post-translational modifications (PTMs) and conformational dynamics. Cartoon representation of eIF2 (α, green; β, yellow; and γ, red), Ser51 is highlighted (orange). Locations of PTMs (phosphorylation and acetylation) are indicated (blue; see also Figure 4b). Interactions stabilised by PTMs are highlighted (blue). GTP-bound eIF2 is highly flexible in solution. After binding of Met-tRNA_i^Met, the ternary complex adopts a more rigid conformation. The unstructured N-terminus of GTP-bound eIF2β interacts with eIF5. Met-tRNA_i^Met release from eIF2 and transfer to the ribosome is triggered by GTP hydrolysis. GDP is exchanged by nucleotide exchange factor eIF2B preparing eIF2 for a new round of translation initiation. Nucleotide exchange is inhibited by phosphorylation of Ser51.