eIF2B is a decameric guanine nucleotide exchange factor with a γ2ε2 tetrameric core.
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.
1] Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK [2] MRC Laboratory of Molecular Biology, University of Cambridge, Francis Crick Avenue, Cambridge CB2 0QH, UK [3].
Yuliya
Y
Gordiyenko
1] Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK [2].
Carla
C
Schmidt
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Martin D
MD
Jennings
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Dijana
D
Matak-Vinkovic
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Graham D
GD
Pavitt
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Carol V
CV
Robinson
D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents; D040681:Structural Homology, Protein
D003432:Cross-Linking Reagents; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D006160:Guanosine Triphosphate