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Abstract
eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (α–ε), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic γ- and ε-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2Bγ, prompting us to propose a multi-step mechanism for nucleotide exchange.
Highlights
EIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2
This results in the dissociation of eIF2-GDP and other associated eIFs to permit 60S joining and transition to the elongation phase2. eIF2-GDP is released from the ribosome in complex with eIF5, preventing the release of eIF2-bound GDP4 until eIF2B displaces eIF5. eIF2B acts as a guanine nucleotide exchange factor (GEF) catalyzing exchange of GDP for GTP, reactivating eIF2 and allowing subsequent rounds of translation initiation
It is generally accepted that eIF2B is composed of five non-identical subunits in unit stoichiometry in yeast[19] and human[28], we show that this complex exists as a decamer in 2:2:2:2:2 stoichiometry in line with very recent results for human eIF2B42
Summary
EIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Hydrolysis of eIF2-bound GTP and Pi release is triggered by correct basepairing of the Met-tRNAi anticodon with the AUG codon of mRNA. This results in the dissociation of eIF2-GDP and other associated eIFs to permit 60S joining and transition to the elongation phase. Diminished eIF2 phosphorylation response causes diseases in mammals ranging from metabolic disorders, to altered sensitivity to viral infection or altered brain functions, including enhanced long-term memory[1] Clinical interest in these proteins has been raised because missense mutations within eIF2 and eIF2B subunits have been linked to distinct inherited neurodegenerative disorders[11,12]. No high-resolution structures are available for the remaining eIF2B subunits, for interactions between eIF2B subunits or between eIF2B and its substrate eIF2
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