CRYSTAL STRUCTURE OF CATALYTIC DOMAIN OF THE INITIATION FACTOR 2B EPSILON SUBUNIT Thomas Boesen1,Sarah S. Mohammad2, Graham Pavitt2, and Gregers R. Andersen1* 1Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Århus C, Denmark 2Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester, M60 1QD, UK Eukaryotic initiation factor 2B (eIF2B) is the exchange factor of initiation factor 2 (eIF2) and catalyses the reaction where GDP bound to eIF2 is exchanged for GTP, a crucial step in translation. The crystal structure of the C-terminal catalytic domain of the eIF2Bε subunit, residues 544-704, from Saccharomyces cerevisiae has been determined to 2.3 Å resolution by the multiple wavelength anomalous dispersion technique using selenomethionine substituted protein. The structure consists of eight α-helices arranged in a manner similar to HEAT repeats. The N-terminal two helices contain the catalytic part of the domain, whereas the C-terminal six helices harbor the two Aromatic Acidic (AA) box motifs. This motif is also found in initiation factor 5, the GTPase activator protein of eIF2, and furthermore in mammalian initiation factor 4G. In eIF2B and eIF5 this motif is involved in binding to the N-terminal part of the eIF2β subunit The aliphatic residues in the AA box motifs are involved in specific contacts in the hydrophobic core of the C-terminal helices important for maintaining the overall structure, whereas acidic residues in the motifs form a clustered, surface exposed acidic patch which might interact with the lysine boxes of eIF2β. Interestingly, Tryptophan 699 was found to be solvent exposed and involved in crystal packing. This residue could possibly be important for the specific interaction with eIF2β. Furthermore, the structure shows the location of residues important for catalytic function and the location of residues for which mutations in humans give rise the the fatal brain disorder leukoencephalopathy with vanishing white matter.