Isoaspartate (isoAsp) forms when asparagine or aspartate in a protein spontaneously rearranges through an aspartimide intermediate, inserting an extra methylene group into the peptide backbone. This modification is usually considered damage associated with aging, and it accumulates in long-lived proteins such as the crystallins of the eye lens. Yet in the ribosomal protein uS11, isoaspartate is present stoichiometrically in bacteria, archaea, and eukaryotes, pointing to a functional role. The paradox is that E. coli doubles in about 20 minutes, while the spontaneous conversion of asparagine to isoaspartate takes days. So there must be an enzyme that accelerates this reaction.
To find a candidate, the authors used AlphaFold3 to predict complexes of uS11 with all 4403 proteins of the E. coli proteome. Of all these proteins, only three gave a confident binding model, and only YbeY placed asparagine-119 of uS11 in a catalytically competent position next to a zinc ion. Then YbeY and uS11 were expressed and purified, and isoaspartate formation was detected by mass spectrometry after trypsinolysis. To test the mechanism, the authors used mutant variants of YbeY, isothermal titration calorimetry to measure zinc binding, and NMR titration of the complex from Thermotoga maritima. A phylogenetic analysis of 189,801 bacterial genomes from the GTDB database linked the presence of YbeY to the asparagine-glycine motif in uS11.
In vitro, E. coli YbeY converts almost all uS11 into the isoaspartate form at position 119 within one hour. The reaction depends on zinc: adding EDTA partially inhibits it, and the H114A mutation, which disrupts zinc coordination, reduces activity to 50%. Mutations of residues involved in substrate binding (T42, R44, D85) also weaken catalysis, whereas the R59A substitution, important for YbeY's RNase activity, does not affect isoaspartate formation. This points to a separate active site for protein modification. Phylogenetics showed that the loss of YbeY in bacterial lineages is accompanied by replacement of asparagine-119 with glycine or by truncation of uS11. In archaea and eukaryotes, the role of YbeY is probably performed by the ATPase Fap7: in the crystal structure of the Fap7–uS11 complex from Pyrococcus abyssi, the authors found that residue 124 is actually isoaspartate, not aspartate as previously thought. Cryo-electron microscopy of ribosomes from an E. coli strain with ybeY deleted showed that the absence of the modification does not change the folding of uS11 but disrupts binding of the ribosomal protein bS21: its density in the map decreases, and the contact between arginine-35 of bS21 and isoaspartate-119 of uS11 disappears. Ribosomes without YbeY associate less well into 70S particles and translate a reporter protein at only 30% of normal.
The work turns isoaspartate from a damage marker into a regulated post-translational modification important for ribosome assembly. The discovery of two independent enzymatic pathways (YbeY in bacteria and Fap7 in archaea and eukaryotes) shows that this modification arose in evolution at least twice. The link between isoaspartate in uS11 and retention of the bS21 protein explains why mutations that disrupt this modification lead to translation defects and slower growth.
It remains to be determined exactly how YbeY coordinates zinc and substrate in the transition state, and to confirm the proposed mechanism via an aspartimide intermediate. For Fap7, it must be proven that it actually phosphorylates aspartate and catalyzes isomerization, rather than simply binding uS11. It would also be interesting to check whether other proteins are modified by YbeY, and how isoaspartate in uS11 affects ribosome dynamics during translation.
The results matter for the molecular biology of translation, the structural biology of the ribosome, and our understanding of post-translational modifications. They may also be useful in developing antibiotics targeting YbeY, since this enzyme is conserved in bacteria but absent from the human cytosol.
Determine the crystal or cryo-EM structure of the YbeY–uS11 complex in a catalytically active state to see zinc coordination and substrate orientation. Test Fap7 activity in vitro using purified proteins and ATP. Investigate which other proteins may be substrates of YbeY in the cell.
The work connects two fundamental problems of molecular biology: the mechanism of ribosome assembly and the role of post-translational modifications in that process. It shows that isoaspartate, usually regarded as spontaneous damage, can be created enzymatically to perform a structural function. This changes how we view the evolution of ribosomal proteins and how cells control the accuracy of translation.
The spontaneous conversion of asparagine to isoaspartate in a peptide with the Asn-Gly motif has a half-life of about 1.2 days, but YbeY speeds this reaction up to minutes, which is necessary for assembling the ribosome within the E. coli cell cycle (about 20 minutes).
Not published on arXiv. Here are its page, the PDF and the formalization: you can check it yourself, not only read our account.