Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Abstract

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA(Gln) formation. To analyze the peculiarities of the structure-function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3 A and a docking model of the synthetase complexed to tRNA(Gln) constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA(Gln) are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA(Gln) recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA(Gln) acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.

Figure 1.

Comparative alignments of Dr GlnRS with bacterial GlnRSs and of related bacterial GatB with Yqey proteins and schematic description of the modular organization of these proteins. Panel (A): Structure-based sequence alignment of GlnRSs cores from E. coli, T. thermophilus and D. radiodurans (prepared with Jalview (37) and based on an alignment including 49 bacterial sequences). Residues are colored using the ClustalX scheme—orange: glycine (G); yellow: proline (P); blue: small and hydrophobic amino acids (A, V, L, I, M, F, W); green: hydroxyl and amide amino acids (S, T, N, Q); red: charged amino acids (D, E, R, K); cyan: histidine (H) and tyrosine (Y)—correspond to positions showing more than 40% identity. Secondary structure elements of Dr GlnRS determined using DSSP (38) are displayed below the sequences on a background using the following code to highlight the structural modules of the GlnRS core: from N- to C-terminus, dinucleotide fold (DNF) in dark blue, acceptor-stem binding domain (ABD) in light blue, helical subdomain (HSD) in green, distal and proximal beta-barrels (DBB and PBB) in magenta and violet, respectively. Residues interacting in E. coli binary and ternary complexes (1O0B, 1GTR) with ATP/AMP, Gln/5′O[N-(l-Gln)sulfamoyl] adenosine and tRNA are indicated by A (a), Q (q) or R (r), respectively (lower case letters are for main-chain interactions, upper case letters for side-chain contacts) and hQ stands for side-chain interactions with glutamine mediated by H₂O molecules. The regions of the enzyme that are not observed in the electron density map are underlined by black lines. The two long insertions of Dr GlnRS are highlighted and class I motives HL/IGH and T/MSK are boxed. Panel (B): Schematic organization of the three representative architectures of the Yqey family (PFAM 02637). For GlnRS, ASD and ACD stand for active side domain and anticodon domain, respectively, with structural modules abbreviated as indicated in (A); for GatB, (B_N) and (B_C) are the N- and C-terminal regions, respectively. Panel (C): Alignement of the C-terminus of Dr GlnRS and other members of the Yqey family (based on the PFAM description of this α-helical domain (PFAM 02637)). Residues displaying at least 40% identity are highlighted with color code as in (B). The secondary structure of B. subtilis Yqey (PDB id: 1NG6) is indicated.

Figure 2.

Analysis of the structural relationship between Yqey domains of Dr GlnRS and bacterial GatB by Western - blots. (A) Western-blot analyses using antibodies directed against T. thermophilus GatCAB AdT or Dr GlnRS. The proteins analyzed are indicated on the top of the lanes. (B) Schematic representation of the full length and deletion mutants of Dr GlnRS used in this analysis. The nature and the position of the last or the first amino acids of the different GlnRS variants are indicated on the basis of the amino acid numbering of FL-GlnRS; in panel (B) tRNA-synt_1c_N and tRNA-synt_1c_C indicate the N- and C-terminal regions of the class Ic GlnRS and Yqey, the Yqey domain.

Figure 3.

Comparison of Ec and Dr GlnRSs. (A) Ec GlnRS in complex with cognate tRNA, Gln and AMPCPP (40) (overall structure colored in gold) is superimposed to the visible core of Dr GlnRS (this study). Domains of the latter are depicted in distinct colors and their definition follows the nomenclature of the E. coli system (41). Color code from N- to C-terminus: dinucleotide fold in dark blue, acceptor-stem binding domain in light blue, helical subdomain in green, proximal and distal beta-barrels in violet and magenta, respectively. (B) This picture of Dr GlnRS highlights in orange and red all common positions that are superimposed with a distance lower than 3.5 Å. In contrast, white patches reveal the regions which are structurally different. Regions with strictly conserved amino acids are in red.

Figure 4.

Analysis of the binding of tRNA^Gln(UUG) to FL-GlnRS (A), Δ-GlnRS (B) and FL-Yqey domain with or without Δ-GlnRS (C) by gel retardation and determination of the K_D of the FL-GlnRS·tRNA^Gln(UUG) complex (D). Gel shifts were performed with 5 × 10⁵ cpm of [³²P]-labeled tRNA^Gln(UUG) transcript and 0–10 µM of FL-GlnRS or 0–15 µM Δ-GlnRS or 0–50 µM FL-Yqey or 0–50 µM Δ-GlnRS and FL-Yqey.

Figure 5.

Model of D. radiodurans GlnRS·tRNA^Gln complex. Residues of the GlnRS core described in the X-ray structure are depicted with the same color code as in Figure 3. Amino acids that are not observed in the experimental electron density map have been modeled and are shown in orange. The Dr GlnRS structure was perturbed using a normal mode analysis in order to fit the conformation with that of the complexed Ec GlnRS and to dock the tRNA as observed in the Ec complex. N and C refer to the N- and C-terminal ends of the protein. The model illustrates the unusual length and flexibility of the N-terminal tail, the large PBB loop close to the active site and the C-terminal domain. The Yqey-like appendix is located at the end of a 60–80 residues long linker and can potentially interact with any part of the tRNA or of the GlnRS because of its flexibility highlighted by the arrows.