Avalilação PREreview de Universally conserved isoasparylation sustains ribosome biogenesis and function

Overall assessment:

This study presents strong evidence that two proteins, YbeY (bacteria and organelles) and Fap7 (archaea, eukaryotes), are involved in the installation of isoaspartate in ribosomal protein uS11. Through bioinformatic analysis, biochemical assays, structural and mutational studies, this work demonstrates the generation and role of the universally-conserved isoaspartate residue in proper assembly and function of the ribosome. The main findings of the study are 1. The role of YbeY and Fap7 in modification of Asn or Asp residues to isoaspartate in the ribosomal protein uS11 across the tree of life. 2. The catalytic mechanisms of isoaspartylation that are novel and highlights mechanistic repurposing of protein folds. While YbeY shares structural and functional similarity with the metzincin metalloprotease in substrate engagement and α-peptide bond hydrolysis, the former keeps the C-terminal tail intact through the β-peptide bond of isoAsp. This mechanistic repurposing of a protein-fold is also accentuated by its eukaryotic counterpart Fap7 where an adenylate kinase fold, typically used for nucleotide phosphorylation, has been repurposed for phosphate transfer from ATP to the target Asp followed by backbone cyclization to form the aspartimide intermediate and subsequent hydrolysis to generate the isoAsp residue. 3. The importance of YbeY or Fap7-mediated modification in correct incorporation of the small ribosomal subunit. 4. The portability of the two isoaspartylation pathways between bacteria and archaea. 5. Providing molecular explanations for previous genetic data showing the importance of YbeY/Fap7 and their mutants.

The study is an important advancement in our understanding of this universal isoaspartylation in proper ribosome functioning. Earlier studies of universally conserved ribosome modifications focused on rRNA, and isoAsp was mainly known in other proteins as an accumulated ageing modification. Therefore, this work addresses a gap in the literature related to translation-related protein modifications.

Based on the experiments conducted to support the claims, I have the following concerns and suggestions which may improve the clarity of this study.

Major comments:

1. In Fig. 1B, the authors have conducted an extensive bioinformatic analysis of uS11 proteins of bacteria and organelle uS11 where species encoding for the YbeY protein predominantly contain an Asn residue at the C-terminal of uS11 which is ‘mostly’ absent in species that do not encode YbeY. However, Fig. 1B also shows a small proportion of an Asn residue at the modification site present in some species that also lack the ybeY gene. A short analysis of this subset would be helpful. i) Do the species that still contain the Asn in uS11 but do not code of YbeY also lack the modification? If so, it may be an evolutionary remnant. ii) If the residue is modified, is there a homologous protein that may carry out a similar function? Because the authors suggest that the isoaspartylation is universally conserved, this explanation would provide further clarity to this statement.

2. The authors show in their structural studies an extensive interaction between uS11 and the modifying enzyme. Yet, the successful transformation of the short peptide fragment (derived from the C-terminal tail of human uS11, Fig 3 and S7) by Fap7 suggests that such extensive interactions are not essential. This suggests that the modification may happen either on the C-terminal tail of the folded protein or on the nascent polypeptide chain. The authors may wish to discuss this.

3. In context of the information shown in Fig. S6, the Zn-free aspartimide could have formed during co-expression, sample preparation or crystallization. As I understand, hydroxylamine can weakly chelate divalent metal ions. At alkaline pH, hydroxylamine can cleave Asn-Gly bonds (as in this case; Fig S6F) which results in succinimide formation (reference 35). This product is the same C-terminal hydroxyamide that the authors detect in Fig S6F. While the zinc loss does not change the interpretation of the results by itself, the current presentation suggests the Zn is not involved in aspartimide generation and this conclusion may not be correct. The Zn-containing sample has already converted Asn119 to isoAsp. The comparison therefore cannot distinguish between a pre-existing aspartimide from one formed during treatment. Perhaps the authors would like to clarify this uncertainty.

4. How do the authors envision Fap7 catalyzing the regioselective hydrolysis of the aspartimide? For YbeY, the Zn provides a clear avenue for regioselective hydrolysis (Fig. S6I), but in Fap7, this is less clear. PIMTs usually result in non-selective hydrolysis as also cited by the authors in reference 33. The authors may wish to discuss this, for example in light of the Lys106, Tyr102 and Pro9 interactions with the product (Fig. 3E). It appears that the Lys106 and Tyr102 anchor the α-carboxyl of isoAsp124 while the Pro9 stabilizes the methylene.

Minor comments

5. ‘YbeY and Fap7 evolutionarily co-occur with uS11 isoaspartylation signatures’ section: The main text states 5551 genomes were analysed (P3 line18), but Fig. 1B panel shows (5191 + 571) genomes in the plot. Perhaps the authors can address this discrepancy in the figure legend and the methods section. Were the organelle genomes counted separately from the bacterial genomes?

6. The authors have demonstrated that the C-terminal tail of uS11 interacts with the active site of YbeY and Fap7 and that the C-terminal peptide fragment alone can act as a substrate. However, the main text and legend of Fig. 3F are missing the sequence details of corresponding peptide fragments used for in vitro testing. The methods section (Table S9) mentions which peptide substrate was used but perhaps naming them and mentioning the sequence in the main text or the figure legends will provide better readability. The same is true for Fig S3B where capillary electrophoresis was used to distinguish Asp and isoAsp containing C-terminal peptide but the peptide used is not mentioned.

7. Fig S7B legend states that reaction with human uS11 peptide was performed for 1 hr at 37 C but the methods state the reaction was conducted for 50 min. The authors may want to address this minor discrepancy.

8. P 3 line 29: the authors may wish to clarify that the sequence dependence is for non-enzymatic isoAsp formation. Bacterial PIMT enzymes generate isoAsp/aspartimide in a variety of sequence contexts (e.g. Refs 50, 51 and Zhu and Link, 2026, Biochemistry, 65(6), 661–669, DOI: 10.1021/acs.biochem.6c00055. PMID: 41790433)

9. P 3 Line 42: The statement that PIMT ‘specifically recognizes and methylates isoAsp residues’ may be too strong, since recent studies have shown that PIMT can also methylate C-terminal Asp/Glu (a review: Zhu and Link, 2026, Biochemistry, 65(6), 661–669, DOI: 10.1021/acs.biochem.6c00055). This is also mentioned in the discussion (ref 52, 53). Also, the methods cite ref. 26 for the purification of PIMT, but ref. 26 does not contain the information.

10. The authors mention that Arg44 π-π stacks with Phe27 in two sections (P 4, line 27 and SI, Fig. S4 legend). It seems that cation-π interaction is the more appropriate interaction happening here since Arg lacks an aromatic π-system.

I reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.