A chiral selectivity relaxed paralog of DTD for proofreading tRNA mischarging in Animalia

Abstract

D-aminoacyl-tRNA deacylase (DTD), a bacterial/eukaryotic trans-editing factor, removes D-amino acids mischarged on tRNAs and achiral glycine mischarged on tRNA^Ala. An invariant cross-subunit Gly-cisPro motif forms the mechanistic basis of L-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, named ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a "gain of function" through L-chiral selectivity in ATD resulting in the clearing of L-alanine mischarged on tRNA^Thr(G4•U69) by eukaryotic AlaRS. The proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNA^Thr(G4•U69) genes are in strict association with the presence of ATD, underlining the mandatory requirement of a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.

Fig. 1

ATD is a variant of DTD. a Multiple sequence alignment showing similar but distinct and characteristic sequence motifs in DTD and ATD (motifs 1 and 2). The highly conserved arginine in DTD (Arg7, EcDTD) is indicated by a star above, whereas the invariant arginine in ATD (Arg151, MmATD) is highlighted by a star below. b Phylogenetic classification of DTD and ATD showing their grouping into two separate categories. c Crystal structure of MmATD homodimer (blue) superimposed on that of PfDTD homodimer (cyan; PDB id: 4NBI)

Fig. 2

ATD has similar active site features as compared to DTD’s. a Crystal structures of PfDTD (PDB id: 4NBI) and MmATD showing that motifs 1 and 2 form the active site at the dimeric interface in both. b Structural superposition of MmATD on PfDTD displaying the cross-subunit Gly-Pro motif in both, i.e., the motif from one monomer inserted into the active site of the other monomer. Residues from the dimeric counterpart are indicated by *

Fig. 3

ATD has a Gly-transPro motif in the active site, unlike a Gly-cisPro motif in DTD. a Comparison between Gly-transPro motif in MmATD and Gly-cisPro motif in PfDTD (PDB id: 4NBI) after structural superposition of the two proteins. b The comparison shown in a depicted from a different angle, highlighting the opposite orientation of Gly-Pro carbonyl oxygens of the two proteins. c Ramachandran plot of glycine and proline residues of the Gly-Pro motif of all the available crystal structures of DTD (blue) and ATD (red), highlighting the change of ~180° in the ψ torsion angle. d Interaction of the side chain of Arg7 with the Gly-cisPro motif of the same monomer in PfDTD (PDB id: 4NBI), and of the side chain of Arg151 with the Gly-transPro motif of the dimeric counterpart in MmATD. Residues from the dimeric counterpart are indicated by *

Fig. 4

ATD displays relaxation of substrate chiral specificity. a–d Deacylation of d-Tyr-tRNA^Tyr, l-Tyr-tRNA^Tyr, Gly-tRNA^Gly and l-Ala-tRNA^Ala by different concentrations of MmATD. Aminoacyl-tRNAs were used at 200 nM final concentration. Error bars denote one standard deviation from the mean of three independent readings

Fig. 5

Proofreading of L-Ala-tRNA^Thr(G4•U69) by ATD is conserved across organisms. Deacylation of l-Ala-tRNA^Thr(G4•U69) and l-Thr-tRNA^Thr(G4•U69) by different concentrations of a MmATD, b HsATD, c GgATD, and d DrATD. Aminoacyl-tRNAs were used at 200 nM final concentration. Er`ror bars denote one standard deviation from the mean of three independent readings

Fig. 6

EF-Tu confers protection on L-Thr-tRNA^Thr(G4•U69) against ATD. Deacylation of l-Ala-tRNA^Thr(G4•U69) by different concentrations of MmATD in the presence of a unactivated EF-Tu, and b activated EF-Tu. Deacylation of l-Thr-tRNA^Thr(G4•U69) by different concentrations of MmATD in the presence of c unactivated EF-Tu, and d activated EF-Tu. Aminoacyl-tRNAs and EF-Tu were used at 200 nM and 2 µM final concentration, respectively. Error bars denote one standard deviation from the mean of three independent readings

Fig. 7

Enrichment of tRNA^Thr(G4•U69) genes and presence of ATD show strict association. a Distribution of AlaRS^ND, tRNA^Thr(G4•U69) genes, and ATD in different domains of life. tRNA gene sequences of Cnidaria and Mollusca are not available in the database. b Bar graph (logarithmic scale) depicting genome size, total number of tRNA^Thr genes, and number of tRNA^Thr(G4•U69) genes occurring in representative organisms belonging to all the three domains of life. Inset showing the number of total tRNA^Thr genes and tRNA^Thr(G4•U69) genes in normal scale; genome size has not been shown for the sake of clarity. Presence of ATD is highlighted in light blue box. Data for occurrence of AlaRS^ND and tRNA^Thr(G4•U69) genes have been taken from reference . c Bar graph showing the number of organisms containing (G4•U69)-harboring tRNA genes which code for tRNAs specific for various proteinogenic amino acids

Fig. 8

ATD is a unique and dedicated proofreading factor that rectifies a critical tRNA selection error. Model for mis-selection and consequent misacylation of tRNA^Thr(G4•U69) with l-alanine by AlaRS^ND, and its subsequent proofreading by ATD. Cognate and non-cognate tRNAs (clover leaf model) are colored in green and red, respectively. Likewise, cognate and non-cognate amino acids (circle) are rendered in green and red, respectively