Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the α-subunit of the only translation initiation factor (eIF2α) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an α + β sandwich fold consisting of two layers: a four-stranded antiparallel β-sheet, and three side-by-side α-helices, with an αββββα α topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive β-turns that overlap with each other by two residues (triple β-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in α-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting β-strand 4 and α-helix 3. The structural architecture, including the triple β-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.