Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes

Abstract

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 A, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors.

Figure 1

The crystal structure of the MetRS-N–Arc1p-N complex reveals domain swapping between symmetry related molecules of MetRS-N. The N-terminal 55 amino acids, comprising most of the β-sheet and the first α-helix, are exchanged between neighboring molecules related by a true crystallographic 2-fold axis. Monomers of MetRS-N are shown in yellow and blue, Arc1p-N is shown in green. The red dotted lines indicate how the putative monomeric MetRS-N was modeled. See text for explanation and discussion.

Figure 2

Multiple structure based sequence alignment of Arc1p-N, MetRS-N and GluRS-N with their most similar structures reported by DALI. Shown are the top 12 DALI hits (labeled by their pdb accessions, supplementary Table 1) structurally aligned with Arc1p-N, MetRS-N and GluRS-N shaded by conservation at three levels. Residues labeled (*) in the line termed ‘core’ are part of the structural alignment core. Secondary structure elements of the classical GST fold are indicated via S for β-sheets and H for α-helices in the line termed ‘2-D’ and represented graphically above the alignment. Residues of MetRS-N interacting with Arc1p-N are indicated by blue dots (in line ResM), residues of Arc1p-N interacting with MetRS-N by green dots (in line ResA), residues of Arc1p-N interacting with GluRS-N by yellow dots (inline ResA), residues of GluRS-N interacting with Arc1p-N by red dots (in line ResG) and contacting residues of the E.coli GST homo-dimer (Pdb accession 1A0F) are indicated by black dots (in line Res1).

Figure 3

The architecture of the Arc1p-N–MetRS-N complex corresponds to the classical GST homo-dimer while Arc1p-N and GluRS-N interact in a novel way. (A) E.coli GST homo-dimer (gray, pdb accession 1A0F) viewed along the 2-fold rotational symmetry axis. (B) The MetRS-N–Arc1p-N complex viewed in an orientation corresponding to that shown for E.coli GST in (A). Arc1p-N is shown in green, MetRS-N in blue. (C) The GluRS-N–Arc1p-N complex viewed in an orientation corresponding to GluRS-N superposition over the top chain of E.coli GST in (A) and over MetRS-N in (B). Arc1p-N is shown in yellow and GluRS-N in red. Corresponding α-helices of the GST domain are labeled 1–7.

Figure 4

The MetRS-N–Arc1p-N interface. Stereo-view of the MetRS-N (blue)–Arc1p-N (green) complex interface with helix labels as in Figure 3B. Selected contacting residues are shown in stick mode and colored in a linear gradient from white (10%) to green (90%) by sequence conservation among orthologous sequences (Figure 5). The two conserved alanines are labeled A26 (Arc1p) and A63 (MetRS).

Figure 5

Multiple sequence alignment of S.cerevisiae Arc1p-N (A), MetRS-N (B) and GluRS-N (C) with orthologs from other yeasts. Shown are the sequences from Ustilago maydis (Um), Schizosaccharomyces pombe (Sp), Yarrowia lipolytica (Yl), Debaryomyces hansenii (Dh), Candida albicans (Ca), Kluyveromyces lactis (Kl), Ashbya gossypii (Ag) and S.cerevisiae (Sc) shaded by conservation at three levels. Secondary structure elements of Arc1p-N, MetRS-N and GluRS-N are indicated via S for β-sheets and H for α-helices in the line termed ‘2-D’ and represented graphically above the alignment. Contacting residues are color coded as in Figure 2. Sequences correspond to the following GeneBank accessions: U.maydis Arc1p: XP_400509.1, GluRS: XP_402397.1, MetRS: XP_401146.1; S.pombe Arc1p: NP_594656.1, GluRS: NP_593483.1, MetRS: NP_595586.1; Y.lipolytica Arc1p: XP_503499.1, GluRS: XP_504508.1, MetRS: XP_506024.1; D.hansenii Arc1p: XP_456881.1, GluRS: XP_461343.1, MetRS: XP_462423.1; C.albicans Arc1p: XP_713255.1, GluRS: XP_720349.1, MetRS: XP_721864.1; K.lactis Arc1p: XP_455553.1, GluRS: XP_451028.1, MetRS: XP_451421.1; A.gossypii Arc1p: NP_985516.1, GluRS: NP_985811.1, MetRS: NP_986998.1; S.cerevisiae Arc1p: X95481, GluRS: P46655, MetRS: P00958.

Figure 6

The GluRS-N–Arc1p-N interface. Stereo-view of the GluRS-N (red)–Arc1p-N (yellow) complex interface with helix labels as in Figure 3C. Selected contacting residues are shown in stick mode and colored in a linear gradient from white (10%) to green (90%) by sequence conservation among orthologous sequences (Figure 4). The two conserved arginines engaged in a stacking interaction at the center of the interface are labeled R164 (GluRS) and R100 (Arc1p).

Figure 7

Several single amino acid point mutations rationally designed based on the complex crystal structures abolish formation of a stable complex between full-length Arc1p and MetRS or GluRS in solution under near-physiological conditions while they do not interfere with stable association of Arc1p with the other enzyme. (A) Mutants Arc1p(A26R) and MetRS(A63H) interfere with Arc1p-MetRS association (B) Mutants Arc1p(R100A) and GluRS(R164A) interfere with Arc1p-GluRS association. Shown are coomassie stained SDS–PAGE analysis of pull-down assays using as bait 6His-tagged GluRS (G), GluRS(R164A) (G*), MetRS (M) or MetRS(A63H) (M*) bound on Ni-NTA beads with untagged Arc1p (A), Arc1p(A26R) (A* in A) or Arc1p(R100A) (A* in B) as prey proteins.

Figure 8

Model of the GluRS-N–Arc1p-N–MetRS-N ternary complex. Superimposing the Arc1p-N parts of the two binary sub-complexes reveals the structural organization of the ternary complex. (A) The Arc1p-N parts from the MetRS-N–Arc1p-N and GluRS-N–Arc1p-N complexes superpose with an RMSD of 0.67 Å, revealing the structural organization of the ternary complex. Helices involved in the interactions are labeled as in Figure 3 and N-termini (N) and C-termini (C) of each protein are indicated. Residue labels (Arc1p K7, E32 and GluRS-N85) indicate boundaries of the deletion constructs used in. (B) Stereo-view of the GluRS-N (red)–Arc1p-N (yellow) and Arc1p-N (green)–MetRS-N (blue) complexes with their Arc1p-N moieties superposed.

Figure 9

Domains with predicted GST fold are conserved among various components of the eukaryotic translation and aminoacylation machineries and are thought to mediate protein–protein interactions in several cases. GST_N (orange circles) and GST_C (red squares) domains of eukaryotic translation elongation factors, aaRS and aminoacylation cofactors are highlighted, other domains are shown in gray and known interactions are indicated by brackets. Shown are proteins from S.cerevisiae (Sc) and Homo sapiens (Hs).