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

D-aminoacyl-tRNA deacylase (DTD), a bacterial/eukaryotic trans-editing factor, removes d-amino acids mischarged on tRNAs and achiral glycine mischarged on tRNAAla. 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 tRNAThr(G4•U69) by eukaryotic AlaRS. The proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr(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.


Reviewer #2 (Remarks to the Author):
The manuscript by Kuncha et al. reports structural and biochemical characterization of a new group of enzymes involved in maintaining fidelity during proteins synthesis. These enzymes, named ATD, are mostly found in Animalia and share sequence and structural similarity with DTD enzymes that hydrolyze D-aminoacyl-tRNAs. Crystal structures clearly show that a key Gly-Pro dipeptide undergoes a cis-to-trans switch from DTD to ATD, which enlarges the active site and allows recognition and hydrolysis of L-aminoacyl-tRNA by ATD. The authors further show that ATD proofreads L-Ala-tRNAThr misformed by AlaRS, providing a previously unknown mechanism of quality control in protein synthesis. Overall, this work is beautifully done and is suitable for the broad readership of Nature Communications. The conclusions, with a minor exception, are well-supported by the results.
Specific comments: • Comparing structures of ATD and DTD reveals that the position of a key Arg locks Gly-Pro in trans and cis positions, respectively. Has any mutational studies been performed on the Arg residue? Does removing Arg7 allow DTD to hydrolyze L-aa-tRNA, or restoring Arg at the corresponding position abolish ATD's activity towards L-aa-tRNA? • The concentrations of aa-tRNAs should be included in the legends of Figures 4 and 5. • The authors should be able to calculate the apparent deacylation rates from Figures 4 and 5. These rates will be useful to better compare substrate selection by ATD. • Figures 4 and 5, the apparent deacylation rate of MmATD is ~500-fold higher for L-Ala-tRNAThr than for L-Ala-tRNAAla. Is there any structural model to explain how it recognizes the first few pairs of the tRNAThr? • Has it been tested to see if L-Ala-tRNAThr is a substrate for DTD? • Line 230 and 640, it's too strong a statement to claim that EF-Tu does not protect L-Ala-tRNAThr from ATD. If fact, Figures 6A and 6B shows that at 5 nM ATD, EF-Tu shows some protective effect. The authors need to revise the statement and related discussion.

Reviewer #3 (Remarks to the Author):
None Response to reviewers' concerns/comments # Reviewer 1

The authors claim that the error in amino acid selection by aminoacyl-tRNA synthetases
is as high as one in 100-1000, which is reasonable based simply on binding affinity. Yet this doesn't take into account the editing capabilities of the synthetases, as the authors certainly know (and with this work contribute to). Perhaps a more full statement would be that because of the low selectivity, these editing functionalities have evolved (primarily in synthetases and associated trans-acting factors) to achieve the ~100-fold higher accuracy in translation.

Reply:
As suggested by the reviewer, we have now made the necessary change in the revised manuscript.

Reply:
The style of in-text citations has now been standardized in the revised manuscript and conforms to the journal format/guidelines.

The concentrations of aa-tRNAs should be included in the legends of Figures 4 and 5.
Reply: The concentrations of aa-tRNAs and EF-Tu have now been mentioned in the legends of respective Main and Supplementary Figures.

Reply:
We agree with the reviewer that values of apparent deacylation rates provide a better appreciation and comprehension of an enzyme's selectivity towards multiple substrates, and we thank the reviewer for pointing out this useful fact. The following table provides these values, which we have now also included in the revised manuscript (Supplementary Table  2).

Figures 4 and 5, the apparent deacylation rate of MmATD is ~500-fold higher for L-Ala-tRNA Thr than for L-Ala-tRNA Ala . Is there any structural model to explain how it recognizes the first few pairs of the tRNA Thr ?
Reply: As is evident from the aforesaid data, the ~500-fold difference in the activity of MmATD on the two substrates, L-Ala-tRNA Thr and L-Ala-tRNA Ala , is attributed to the differences in the two tRNAs, viz., tRNA Ala and tRNA Thr . Hence, the focus of our future work will be to identify the elements of the tRNA that are involved in this discrimination and the underlying mechanism through biochemical, biophysical and structural investigations, including the determination of ligand-bound crystal structures. This will allow us to propose a structural/mechanistic model for ATD's substrate specificity.

Reply:
We are grateful to the reviewer for bringing up a relevant aspect of DTD with regard to its activity on L-Ala-tRNA Thr . We have now tested DTD's activity on L-Ala-tRNA Thr and we show that the enzyme does not act on the substrate even at 100 nM concentration (Fig. 2) compared to ATD's activity on the same substrate at 1 nM concentration (Fig. 1a). The data have now been included in the revised manuscript (Supplementary Fig. 7).