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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.

Original publication

DOI

10.1038/ncomms4902

Type

Journal article

Journal

Nat Commun

Publication Date

23/05/2014

Volume

5

Keywords

Cross-Linking Reagents, Electrophoresis, Polyacrylamide Gel, Eukaryotic Initiation Factor-2, Eukaryotic Initiation Factor-2B, Guanosine Triphosphate, Hydrophobic and Hydrophilic Interactions, Mass Spectrometry, Models, Molecular, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Protein Subunits, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Solvents, Structural Homology, Protein