The structure of tryptophanyl-tRNA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs

Abstract

The 2.1A crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue alpha-helix. This helix replaces a beta-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this beta-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (alpha(2))(2) homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal alpha-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS.

Figure 1. Sequence alignment of the G. lamblia TrpRS N-terminus with other eukaryotic and archaeal sequences

The Giardia and human TrpRS are structurally homologous beginning at residue 20. In the Giardia structure, residues N-terminal to this point form part of an initial α-helix comprising residues 6–22. In the human TrpRS these residues have been observed instead to form a β-hairpin involved in ATP binding. A triad Val-Trp-Val of hydrophobic residues, shown boxed in the figure, stabilizes association of the hairpin with a Trp residue at the positions equivalent to Arg 117 in the Giardia sequence, and with a highly conserved Phe residue equivalent to Phe 301 in the Giardia sequence. This hydrophobic triad and the corresponding tryptophan are recognizably present in most eukaryotic TrpRS sequences, but are missing from some protozoan homologs.

Figure 2. Crystallographically observed tetramer of G. lamblia TrpRS

(a) The two monomers in the upper half of the figure are crystallographically independent. The view is along the crystallographic 2-fold axis relating the monomers in the upper half of the figure to those in the lower half. The pair of monomers on the left (dark green, light green) and the pair monomers on the right (light blue, dark blue) each form the canonical dimer previously observed for all class Ic aminoacyl-tRNA synthetases. The dimer formed by the two crystallographically independent monomers (dark green, light blue) buries 1380 Ų of accessible surface on the N-terminus of each monomer due to the interlocked α1 helices, plus an additional 160 Ų per monomer due to reciprocal interaction of the α11 helices. Buried surface areas were calculated using PISA (Krissinel and Henrick, 2007). (b) Surface representation of the two crystallographically independent monomers, showing how they reciprocally interdigitate to form a dimer. The view is rotated roughly 90° from the view in (a).

Figure 3. Superposition of human and Giardia TrpRS active sites

The complex of human TrpRS with Trp-AMP (PDB 1r6t; Yang et al., 2003) is shown in yellow. The β1β2 hairpin in the center of the figure is necessary for ATP binding and subsequent tRNA-independent activation of tryptophan to yield Trp-AMP (Otani et al., 2002; Guo et al., 2007). The hairpin is necessarily displaced to allow access by the acceptor arm of tRNA so that the activated tryptophan may be transferred to the tRNA. One monomer of the Giardia TrpRS (green) is superimposed onto the human structure (yellow). The short helix + β1β2 hairpin of the human structure is replaced by a single, longer helix α1 at the N-terminus of the Giardia TrpRS monomer (green). The Giardia helix α1 interlocks with the equivalent helix α1 from a second monomer (blue) in the tetramer, so that the active site is bounded by residues from two contributing monomers of the tetramer.

Figure 4. Model for tRNA binding

(a) tRNA bound to the dimeric human TrpRS (PDB 2dr2). Although two tRNA molecules are bound to each dimer in the crystal structure, for clarity only one is shown here. (b) A crude model for tRNA binding to the Giardia TrpRS tetramer. The human TrpRS:tRNA complex was positioned onto the full Giardia tetramer by superposition of the monomer containing the active site (upper monomer in (a), green monomer in (b)). The fourth monomer of the Giardia structure is omitted for clarity. The acceptor arm of the tRNA molecule extends through the central hole in the upper lobe of the tetramer to enter the active site of the monomer at the upper left.

Figure 4. Model for tRNA binding

(a) tRNA bound to the dimeric human TrpRS (PDB 2dr2). Although two tRNA molecules are bound to each dimer in the crystal structure, for clarity only one is shown here. (b) A crude model for tRNA binding to the Giardia TrpRS tetramer. The human TrpRS:tRNA complex was positioned onto the full Giardia tetramer by superposition of the monomer containing the active site (upper monomer in (a), green monomer in (b)). The fourth monomer of the Giardia structure is omitted for clarity. The acceptor arm of the tRNA molecule extends through the central hole in the upper lobe of the tetramer to enter the active site of the monomer at the upper left.