Mechanism of chiral proofreading during translation of the genetic code

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Mechanism of chiral proofreading during translation of the genetic code

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DOI: http://dx.doi.org/10.7554/eLife.01519Published December 3, 2013 Cite as eLife 2013;2:e01519

Abstract

The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant ‘cross-subunit’ Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD’s ability to act on multiple D-amino acids. The strict chiral specificity elegantly explains how the enriched cellular pool of L-aminoacyl-tRNAs escapes this proofreading step. The study thus provides insights into a fundamental enantioselection process and elucidates a chiral enforcement mechanism with a crucial role in preventing D-amino acid infiltration during the evolution of translational apparatus.

DOI: http://dx.doi.org/10.7554/eLife.01519.001

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Acknowledgements

SA and SBR thank Council of Scientific and Industrial Research (CSIR), India for funding. RS acknowledges funding from Swarnajayanti Fellowship of Department of Science and Technology, India and 12th Five Year Plan Project BSC0113 of CSIR, India.

Decision letter

John Kuriyan, Reviewing editor, Howard Hughes Medical Institute, University of California, Berkeley, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

[Editors’ note: the authors performed additional work to address the concerns raised in the first round of peer review and submitted for further consideration. The two decision letters after peer review are shown below.]

Thank you for choosing to send your work entitled “Mechanism of chiral proofreading during translation of the genetic code” for consideration at eLife. Your full submission has been evaluated by a Senior editor and 2 peer reviewers, and the decision was reached after discussions between the reviewers. We regret to inform you that your work will not be considered further for publication.

In this paper, the X-ray crystal structure of Plasmodium falciparum D-aminoacyl-tRNA deacylase (DTD) in a liganded complex with a non-hydrolysable D-tyrosine analogue, D-tyrosyl-3'-aminoadenosine, is presented. The reviewers recognize that the work is a significant contribution to the field and provides further insight into the catalytic mechanism of this enzyme, the details of which could not be determined from the previously solved apo-structure. The authors use mutagenic studies to demonstrate the crucial catalytic role of the highly conserved Gly-cisPro motif within the enzyme as well as showing structurally the importance of this motif in chiral selection. In addition the findings also indicate that this particular DTD is likely to only act upon aminoacylated-tRNA species when the amino acid is attached to the 3' hydroxyl of the terminal adenine of the tRNA donor molecule. Nevertheless, the review has raised some major concerns that have led to the conclusion that the paper is not suitable for publication by eLife.

1) The major issues arise from placing the present work in the context of what is already known about the enzyme. The crystal structure of DTD has been determined earlier. A model docking the substrate analogs with DTD from different sources have also been obtained. Crystal structures of PfDTD with adenosine and different D amino acids have led to a mechanical model that is contrary to the model suggested here. Conclusions in the present manuscript go too far beyond what the data indicate.

The details of the reaction mechanism are still not completely defined, and so one has to make the hypothesis that the observed binding mode is the relevant one. The statement ‘Previous attempts using several apo and complex structures ... yielded the first cognate ligand-bound structure and provided the structural basis of this fundamental cellular process’ is misleading. It has been shown that D-Tyr-adenosine was not hydrolyzed by DTD, but a D-Tyr-esterified oligonucleotide produced by RNase T1 digestion of D-Tyr-tRNA having a 19mer oligonucleotide, is its substrate (Ferri-Fioni et al., 2001). Therefore, present ligand is also a model system for interpretation of the possible mode of substrate binding and catalytic action.

2) In addition to GlycisPro, there are several other amino acids involved in the interactions with the ligand (Figure 1) and if the true substrate that is much larger were to be bound, there would have been many contacts. The authors spent a lot of effort in highlighting the conserved nature of these two residues and their stereochemistry. How does one rule out other possible conserved interactions even if there are sequence differences in the binding pocket?

3) The binding pocket conformation has been assumed to be very rigid while attempting to illustrate that L-amino acid analogs would be sterically excluded (Figure 2). This has been substantially based on the conserved nature of the GlycisPro motif (Figure 6). In principle, the substrate binding pocket of an enzyme has to have certain amount of plasticity and even if the stereochemistry of a couple of residues is conserved, possible structural changes due to the plasticity associated with other residues can not be ruled out. Indeed, this argument is consistent with the degenerate recognition of diverse D-amino acids in case of the same enzyme (Bhatt et al. (2010)). This is important particularly considering that the true substrate is much bigger than the ligand used here. While it is accepted that the L-amino acids are rejected, one has to be cautious about interpreting the mechanism based on the rigidity of a motif consisting of only two amino acids from the binding pocket.

4) Bhatt et al. (2010) have provided ‘atomic snap shots’ for the catalytic mechanism of DTD based on the crystal structures of several complexes of DTD with many D-amino acids and ADP binding. The model proposed in their case emphasizes substantial plasticity at the binding site and highlights the possible catalytic steps. The authors of the present study reject that model based on two counts: they were not cognate ligand complexes and their binding modes are different. Knowing that D-Tyr-adenosine is also not a true substrate of DTD, better explanation would be required to rule out the earlier described binding modes considering they also involve D-amino acids and adenosine.

[Editors’ note: what now follows is the decision letter after additional work had been performed.]

Thank you for submitting your work entitled “Mechanism of chiral proofreading during translation of the genetic code” for further consideration at eLife. Your revised article has been favorably evaluated by a Senior editor and the original two reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

In this paper, the X-ray crystal structure of Plasmodium falciparum D-aminoacyl-tRNA deacylase (DTD) in a liganded complex with a non-hydrolysable D-tyrosine analogue, D-tyrosyl-3'-aminoadenosine, is presented. The work is a significant contribution to the field and provides further insight into the catalytic mechanism of this enzyme, the details of which could not be determined from the previously solved apo-structure. The authors use mutagenic studies to demonstrate the crucial catalytic role of the highly conserved Gly-cisPro motif within the enzyme as well as showing structurally the importance of this motif in chiral selection. In addition the findings also indicate that this particular DTD is likely to only act upon aminoacylated-tRNA species when the amino acid is attached to the 3' hydroxyl of the terminal adenine of the tRNA donor molecule.

An earlier version of this manuscript had been rejected previously, but the authors have done a good job of responding to the criticisms of the previous version by providing additional data concerning the validity of the binding mode of the substrate analog, and by making changes to the text of the manuscript.

The authors should revise the manuscript taking into account the following points. The paper will not be sent out for review again and the editor will make a final decision based the revisions made to the manuscript. Please respond to all of these points by revising the manuscript appropriately.

1) The authors now describe mutations that indicate that the binding mode they see is relevant to catalysis. They also carry out some mutations around the active site that support nucleotide-mediated catalysis, although this point in not rigorously proven because the data are negative (i.e., mutations do not affect catalysis substantially). Hence, they should be careful to tone down the discussion regarding previously solved structures of DTD with other ligands. The focus should be on what can be definitively concluded from the combination of structures presented here and other earlier structures rather than just over stating apparent ‘failures’ of either. This would allow for better discussion of the different catalytic mechanisms proposed from earlier structures obtained with other ligands and what is proposed to be the case here. Specifically, the Introduction should describe the earlier structural work and place the present work properly in context of the previous work, before moving on to a discussion of the present work.

2) It is very difficult to place the results discussed here in the context of DTD from various species (eukaryotes, prokaryotes, mammals...). The Abstract should state which species is analyzed, and the Introduction should also state more clearly the extent to which DTD is conserved in different species and which ones are analyzed here. It is true that this information is available in the manuscript, but it should be provided up front and in a way that the reader can immediately appreciate.

3) The word “novel” should be deleted from the Abstract. It is not clear in what sense the solution that this enzyme has reached for specificity is “novel” and in any case the novelty should be left for others to judge.

4) In the discussion of the active site mutants, it is stated “The above data clearly demonstrate that none of the protein residues around the scissile bond are involved in catalysis.” Since only negative data are presented, this statement is too strong. Change “clearly demonstrate” to “indicate” or some other appropriate word. Likewise, a little later the paper states “…the above data strongly suggest that the DTD fold is designed to be an RNA-based catalyst in the proofreading reaction”. Again, in the absence of positive data about the mechanism, replace “strongly suggest” by “are consistent with” or some other appropriate wording.

5) Towards the end of the paper it is said “These data, therefore, clearly show the lack of functional relevance of the binding modes proposed earlier...”. This is too strong. How do the authors know that the previously seen binding modes are not intermediates for some part of the process of catalysis? Remove this wording and replace with appropriate text.

6) A little later, it is stated “More importantly, the current study identifies and reveals for the first time the key role of an invariant cross-subunit Gly-cisPro motif in solving....”. Delete “and reveals for the first time”. This work is built on earlier structural work and it is sufficient to say that it identifies a key role for this motif.

7) The paragraph in the Discussion beginning “The study opens up important questions....” should be deleted. It is too speculative, and it is far from certain that the structure can be used to design DTD enzymes to create a “D-amino acid world”.

DOI: http://dx.doi.org/10.7554/eLife.01519.028

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