Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNA(Trp) and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3' end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an alpha-helix of the unique N-terminal domain and the anticodon loop by an alpha-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.

Figure 1

Structure of the hTrpRS–tRNA^Trp–Trp complex. (A) Overall structure of the dimeric hTrpRS–tRNA^Trp–Trp complex. hTrpRS is shown as ribbon with the N-terminal fragment in pink, the catalytic domain in green and the C-terminal domain (also called anticodon binding domain) in gold. For clarity, the second hTrpRS molecule is shown in gray. tRNAs are shown as cyan ribbons and the Trp substrates as ball-and-stick models. (B) A representative SIGMAA-weighted 2F_o–F_c map (1.0 σ contour level) in the anticodon loop region of tRNA^Trp at 3.0 Å resolution. The final coordinates of tRNA^Trp (in yellow) and hTrpRS (in light blue) are shown as ball-and-stick models. (C) Structure-based sequence alignment of hTrpRS with representative TrpRSs from other species. hTrpRS, human TrpRS; yTyrRS, S.cerevisiae TrpRS; aTrpRS, Pyrococcus abyssi TrpRS and bTrpRS, B.stearothermophilus TrpRS. Strictly conserved residues are highlighted in shaded red boxes and conserved are shown in open red boxes. The secondary structure of hTrpRS is placed on top of the alignment [after Yu et al. (12)] and the secondary structure of bTrpRS is placed at the bottom of the alignment (9). The N-terminal 96 residues of hTrpRS do not exist in the structure of the hTrpRS–tRNA–Trp complex.

Figure 1

Structure of the hTrpRS–tRNA^Trp–Trp complex. (A) Overall structure of the dimeric hTrpRS–tRNA^Trp–Trp complex. hTrpRS is shown as ribbon with the N-terminal fragment in pink, the catalytic domain in green and the C-terminal domain (also called anticodon binding domain) in gold. For clarity, the second hTrpRS molecule is shown in gray. tRNAs are shown as cyan ribbons and the Trp substrates as ball-and-stick models. (B) A representative SIGMAA-weighted 2F_o–F_c map (1.0 σ contour level) in the anticodon loop region of tRNA^Trp at 3.0 Å resolution. The final coordinates of tRNA^Trp (in yellow) and hTrpRS (in light blue) are shown as ball-and-stick models. (C) Structure-based sequence alignment of hTrpRS with representative TrpRSs from other species. hTrpRS, human TrpRS; yTyrRS, S.cerevisiae TrpRS; aTrpRS, Pyrococcus abyssi TrpRS and bTrpRS, B.stearothermophilus TrpRS. Strictly conserved residues are highlighted in shaded red boxes and conserved are shown in open red boxes. The secondary structure of hTrpRS is placed on top of the alignment [after Yu et al. (12)] and the secondary structure of bTrpRS is placed at the bottom of the alignment (9). The N-terminal 96 residues of hTrpRS do not exist in the structure of the hTrpRS–tRNA–Trp complex.

Figure 2

Recognition of the tRNA^Trp acceptor arm by hTrpRS. (A) Molecular surface of the hTrpRS–tRNA^Trp–Trp complex showing the interactions between the tRNA acceptor arm and the structural elements of hTrpRS. hTrpRS interacts with the tRNA acceptor arm from the major groove. However, the 3′ end CCA takes a sharp turn to enter into the catalytic active site with a deformed conformation. The bound Trp is shown with a ball-and-stick model. (B) A stereoview showing the interactions of the tRNA acceptor arm with the structural elements of hTrpRS. The tRNA is shown as blue ribbons with the nucleotides of the acceptor arm in yellow. The N-terminal fragment of hTrpRS is shown in purple and helices α6 and α9 of the catalytic domain in green. Residues of hTrpRS involved in recognition of the tRNA acceptor arm are shown with side-chains (in light blue). (C) Recognition of the discriminator base A73 by residues of helix α1′ of the N-terminal fragment. The hydrogen-bonding interactions are indicated by thin red lines. (D) Molecular surface at the catalytic active site showing the interactions of the 3′ end CCA of the tRNA acceptor arm with the surrounding residues of hTrpRS. The 3′ end CCA of the tRNA is in the uncharged form and the 2′-OH group of A76 is positioned about 6 Å away from the α-carbonyl carbon of the Trp (as indicated by arrow).

Figure 3

Recognition of the tRNA^Trp anticodon by hTrpRS. (A) Structure of the tRNA anticodon binding site in the hTrpRS–tRNA^Trp–Trp complex. The tRNA is shown as blue ribbon with the anticodon CCA in yellow. The small helical domain of hTrpRS is shown in gold and the short helix α11 in green. Residues involved in the recognition of the tRNA anticodon are shown with side-chains (in light blue). The hydrogen-bonding interactions are indicated with thin red lines. (B) Structure of the tRNA anticodon binding site in the docking model of the bTrpRS–tRNA^Trp complex. Due to the absence of an equivalent helix α11, the C34 base has less interaction with the protein.

Figure 4

Structure of the catalytic active site of hTrpRS. (A) A stereoview showing the interactions between the bound Trp and the surrounding residues of hTrpRS. The Trp is shown in gold and the residues having close interactions with the substrate are shown with side-chains (in gray). The indole N atom of the Trp is specifically recognized by Tyr159 and Gln194 via hydrogen-bonding interactions (indicated by thin red lines). (B) Structural comparison of hTrpRS in the hTrpRS–tRNA^Trp–Trp complex (in yellow), the hTrpRS–Trp–AMP complex (in pink) (10) and the unliganded hTrpRS (in blue) (12). The superposition is based on the catalytic domain (in gray). The regions displaying major conformational changes are indicated with dotted circles. For clarity, only Trp-AMP in the hTrpRS–Trp–AMP complex is shown as a ball-and-stick model.

Figure 5

Potential functional role of the N-terminal β-hairpin of hTrpRS. (A) Structure of the hTrpRS–Trp–AMP complex (10). The N-terminal HTH motif and β-hairpin are shown in purple and the Trp-AMP as a ball-and-stick model. The disordered region linking the HTH motif and β-hairpin is indicated with a dashed line. (B) Molecular surface of the hTrpRS–Trp–AMP complex showing that the β-hairpin covers on top of the catalytic active site and overlaps with the 3′ end CCA of a docked tRNA model based on the hTrpRS–tRNA^Trp–Trp complex.