eIF2B Mechanisms of Action and Regulation: A Thermodynamic View

Abstract

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor of the GTPase eIF2, which brings the initiator Met-tRNA_i to the ribosome in the form of the eIF2-GTP·Met-tRNA_i ternary complex (TC). The activity of eIF2B is inhibited by phosphorylation of its substrate eIF2 by several stress-induced kinases, which triggers the integrated stress response (ISR). The ISR plays a central role in maintaining homeostasis in the cell under various stress conditions, and its dysregulation is a causative factor in the pathology of a number of neurodegenerative disorders. Over the past three decades, virtually every aspect of eIF2B function has been the subject of uncertainty or controversy: from the catalytic mechanism of nucleotide exchange, to whether eIF2B only catalyzes nucleotide exchange on eIF2 or also promotes binding of Met-tRNA_i to eIF2-GTP to form the TC. Here, we provide the first complete thermodynamic analysis of the process of recycling of eIF2-GDP to the TC. The available evidence leads to the conclusion that eIF2 is channeled from the ribosome (as an eIF5·eIF2-GDP complex) to eIF2B, converted by eIF2B to the TC, which is then channeled back to eIF5 and the ribosome. The system has evolved to be regulated by multiple factors, including post-translational modifications of eIF2, eIF2B, and eIF5, as well as directly by the energy balance in the cell, through the GTP:GDP ratio.

Figure 1

Functions and regulation of eIF2B. eIF2 brings the Met-tRNA_i to the ribosomal translation initiation complex, in the form of the eIF2-GTP·Met-tRNA_i ternary complex (TC). Upon start codon recognition, eIF2 hydrolyzes GTP, and eIF2-GDP is released. eIF2B catalyzes nucleotide exchange and Met-tRNA_i binding to form a new TC. Phosphorylation of the α subunit of eIF2 by several stress-activated kinases turns eIF2-GDP from a substrate into an inhibitor of eIF2B. Inhibition of eIF2B activity causes a decrease in the level of global protein synthesis and at the same time triggers the integrated stress response (ISR), which involves both pro-apoptotic and pro-survival pathways. The ultimate fate of the cell is either restoration of homeostasis or apoptosis, depending on the interplay between pro-survival and pro-apoptotic processes in the cell.

Figure 2

Structural basis for the mechanisms of eIF2B action and regulation. (A) Structure of Schizosaccharomyces pombe eIF2B, viewed from the eIF2α-binding pocket. The individual subunits are labeled and color-coded. Only one each of the eIF2Bα, -β, and -δ subunits are visible. (B) Model for the eIF2B·eIF2-GDP complex in an extended conformation from ref (top). eIF2 subunits are shown as ribbons. The side chain of S51 in eIF2α is colored blue. GDP is colored red. Model of the eIF2B·apo-eIF2 complex in a closed conformation from ref (bottom). Only the position of eIF2α-NTD is changed: it now contacts eIF2α-CTD and is near eIF2γ. The equilibrium between the GDP-bound and apo states of the eIF2B·eIF2 complex is shown schematically on the right.

Figure 3

Thermodynamic description of the eIF2B catalytic cycle. (A) Thermodynamic description of the pathway of eIF2B-catalyzed recycling of eIF2-GDP to the TC and ribosome binding. Solid arrows indicate progression along the path, with steps labeled as follows: (I) displacement of eIF5 from eIF2-GDP by eIF2B, (II) eIF2B-promoted dissociation of GDP from eIF2, (III) binding of GTP to eIF2B-bound apo-eIF2, (IV) binding of Met-tRNA_i to form the TC on eIF2B, and (V) displacement of eIF2B from the TC by eIF5. Dashed arrows represent steps off the main pathway. Red crosses mark steps that can be safely ignored because of either off rates being too slow on the time scale of translation or the equilibrium being shifted too far away from the product. Dissociation constants (K_Ds) are provided for each step and represent either experimentally determined values reported in the literature (black) or calculated (blue) or estimated (red) values based on thermodynamic coupling considerations (see Table 1). The ratios of K_Ds and resulting equilibria for the relevant steps are shown in the table on the right. (B) Thermodynamic description of how phosphorylation blocks catalysis by inhibiting eIF2B-mediated nucleotide exchange. The color and overall scheme are identical to those presented in panel A. K_Ds affected by phosphorylation are underlined. Because the affinity of eIF2B for apo-eIF2(α-P) (reaction intermediate) is the same as that for eIF2(α-P)-GDP (substrate), the enzymatic driving force is removed and eIF2B has no effect on GDP dissociation. (C) Activation energies for nucleotide exchange on eIF2 when free, in the presence of eIF2B, and when phosphorylated.