Solution structure of the RWD domain of the mouse GCN2 protein

Abstract

GCN2 is the ␣-subunit of the only translation initiation factor (eIF2␣) 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 ␣ + ␤ sandwich fold consisting of two layers: a four-stranded antiparallel ␤-sheet, and three side-by-side ␣-helices, with an ␣␤␤␤␤␣␣ topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive ␤-turns that overlap with each other by two residues (triple ␤-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in ␣-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 ␤-strand 4 and ␣-helix 3. The structural architecture, including the triple ␤-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 RWDcontaining proteins.

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

“…1 B and C) (22)(23)(24). This fold was also related to those of the RWD (RING finger-, WD-repeat-, and DEAD-like proteins) domain and ubiquitin-conjugating enzymes (25,26). The fold similarity between Mad1 CTD and the CTDs of Spc25 and Csm1 was unexpected, as they share no obvious sequence similarity.…”

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…Furthermore, 50%-75% of GTAP's amino acid sequences appear in several other nonmammalian proteins. In addition to the UBC region, GTAP also contains a RWD domain [22] and a unique NH 2 -terminal domain, which makes GTAP structurally similar to a newly discovered class of Ub-conjugating enzymes: the class III E2 members of Ub-conjugating enzymes [6]. These domains may be crucial for compartmentalization, protein transport, or interactions with chaperones and/or scaffold proteins [7].…”

“…1C, 2A), identical to a putative Ub-conjugating enzyme gene product both from human and mouse called UBE2Q (NM017582 and NM027315, respectively). The deduced amino acid sequence of both human and murine GTAP revealed a novel NH 2 -terminal motif consisting of glutamine-and proline-rich repeats and a characteristic YPXXXP motif in a structurally conserved RWD domain (RING finger, WD repeats, and yeast DEAD-like helicases containing proteins [22]) observed in many other E2-like enzymes (Fig. 1C, #1, #2).…”

Rwdd1, a Thymus Aging Related Molecule, Is a New Member of the Intrinsically Unstructured Protein Family

“…Similarly, the unfolded regions in GCN2 account for only a small portion of the whole protein, and locate outside the functional domains. Furthermore, the folded structure of the RWD domain of GCN2 has been confirmed by NMR (6). Both RSUME and GCN2 show normal electrophoresis behavior.…”

“…This may be critical for Rwdd1 to remain an unstructured form in cytoplasm before it could interact with certain protein molecule and carry out its function. The structure of the RWD domain of mouse GCN2 had been solved to be of an α-β sandwich form (6). Thus, the RWD region in Rwdd1 might transit from unfolded state to folded state when complexing with a partner.…”