Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase

Abstract

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA(Gln) and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

Figure 1.

Deletion of the N-terminal domain of GLN4 impairs function. (A) Mutants bearing a gln4 mutation in which amino acids 2–210 are deleted are defective in growth at low temperature on YP media containing glucose or glycerol as a carbon source. Serial dilutions of strains with either wild-type GLN4 or gln4(211–809) (marked gln4-ΔN*) integrated at the ade2 locus in the gln4-ΔKanR mutant were grown as indicated. Indicated strains carry CEN plasmids either with or without GLN4. (B) Mutants bearing a gln4 mutation in which amino acids 2–210 are deleted are sensitive to the glutamine synthase inhibitor l-methionine sulfoximine (MSX).

Figure 2.

The N-terminal domain of Gln4 is required for specific binding to native tRNA^Gln(CUG). (A) Gln4 variant proteins deleted for different amounts of the NTD exhibit reduced tRNA^Gln(CUG) binding. (B) Gln4(187–809) protein exhibits detectable binding to tRNA^Gln(CUG) at high concentrations.

Figure 3.

Structure of Gln4(1–187) with comparisons to domains in S. aureus GatB (PDB ID: 3IP4). (A) Crystallographic structure of Gln4 residues 1–187 in cartoon representation. The proposed hinge region (Gly₁₁₂Val₁₁₃Gly₁₁₄) is highlighted together with the likely interacting residue Trp₁₆₀, and shown in stick representation. (B) Surface electrostatic model of Gln4 residues 1–187, shown with two orientations rotated by 90° relative to each other, with positively charged residues colored blue. (C and D) Structural alignment of helical and tail domains of Gln4 NTD and S. aureus GatB (PDB ID: 3IP4) (45). (C) The crystal structure of Gln4(1–110) (red) is superposed to the helical domain of GatB(295–406) (cyan). (D) The crystal structure of Gln4(119–178) (red) is superposed on the tail domain of GatB(414–475) (cyan).

Figure 4.

The linker between the two domains in Gln4(1–187) likely behaves as a hinge, is highly conserved and is important for tRNA binding. (A) Structure of Gln4(1–187) (red) superposed on TMGatB (light gray) and SAGatB (dark gray) by alignment of the tail domains. (B) Conservation of GlnRS NTD sequences, red-≥90%; blue-≥70%, with arrow at Gln4₁₈₇., aligned using Multialin (49). (C) Conserved residues are highlighted on Gln4(1–187) according to the color code in B with the NTD backbone shown in light grey. (D) Close contacts between W160 of the Gln4 NTD and other residues.

Figure 5.

Mutations in conserved amino acids in the putative hinge of the NTD affect the interaction of Gln4 with native tRNA^Gln(CUG). (A and B) EMSA wild-type and mutant Gln4 proteins (23–2017 nM). (C) Binding as a function of Gln4 protein concentration.