A metal ion mediated functional dichotomy encodes plasticity during translation quality control

Gogoi, Jotin; Pawar, Komal Ishwar; Sivakumar, Koushick; Bhatnagar, Akshay; Suma, Katta; Ann, Kezia J.; Pottabathini, Sambhavi; Kruparani, Shobha P.; Sankaranarayanan, Rajan

doi:10.1038/s41467-025-58787-2

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Published: 16 April 2025

A metal ion mediated functional dichotomy encodes plasticity during translation quality control

Nature Communications volume 16, Article number: 3625 (2025) Cite this article

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Abstract

Proofreading during translation of the genetic code is a key process for not only translation quality control but also for its modulation under stress conditions to provide fitness advantage. A major class of proofreading modules represented by editing domains of alanyl-tRNA synthetase (AlaRS-Ed) and threonyl-tRNA synthetase (ThrRS-Ed) features a common fold and an invariant Zn²⁺ binding motif across life forms. Here, we reveal the structural basis and functional consequence along with the necessity for their operational dichotomy, i.e., the metal ion is ubiquitous in one and inhibitor for the other. The universally conserved Zn²⁺ in AlaRS-Ed protects its proofreading activity from reactive oxygen species (ROS) to maintain high fidelity Ala-codons translation, necessary for cell survival. On the other hand, mistranslation of Thr-codons is well tolerated by the cells, thereby allowing for a ROS-based modulation of ThrRS-Ed’s activity. A single residue rooted over ~3.5 billion years of evolution has been shown to be primarily responsible for the functional divergence. The study presents a remarkable example of how protein quality control is integrated with redox signalling through leveraging the tunability of metal binding sites from the time of last universal common ancestor (LUCA).

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Introduction

It is a general notion that translation of the genetic code proceeds with a high level of accuracy^1,2. However, there is increasing evidence that a compromise in translation quality control and the consequent mistranslation could serve beneficial roles and evolve under positive selection^{3,4,5,6,7,8,9}. Growing body of work has now established that adaptive misaminoacylation of tRNAs by aminoacyl-tRNA synthetases (aaRSs) can give rise to beneficial mistranslation through generation of non-cognate aminoacyl-tRNA (aa-tRNA) pairs^{8,10,11,12,13}. However, not all tRNA mischarging events have beneficial prospects as a majority of them are detrimental to the cell^2,14,15,16. One of the intriguing instances of this dichotomy in the effect of mischarging is noted in connection with two universally conserved aaRSs: alanyl-tRNA synthetase (AlaRS) and threonyl-tRNA synthetase (ThrRS).

AlaRS and ThrRS belong to the same sub-group of aaRSs – class II, owing to the homology of the aminoacylation domain¹⁷. Both AlaRS and ThrRS, apart from charging their cognate amino acid, L-alanine and L-threonine respectively, also transiently mischarges similar non-cognate amino acids: AlaRS mischarges glycine and L-serine, whereas ThrRS mischarges L-serine^18,19,20. A universally conserved editing domain, distinct from the aminoacylation domain, was identified in AlaRS (AlaRS cis-Ed)²¹ and was shown to proofread Ser/Gly mischarged on -tRNA^Ala ²². Remarkably, apart from the editing domain appended to AlaRS, multiple trans-editing AlaRS cis-Ed homologues called as AlaXps were identified viz., AlaX-L, AlaX-M and AlaX-S (Supplementary Fig. 1a)^23,24, which together constitute AlaX-like family (SCOP id: 4001430) (From here on, AlaRS-Ed will refer to all the homologues of AlaX-like family). Indeed, AlaRS-Ed represents one of the most redundant and yet most conserved editing domain in the context of translation quality control, deployed as multiple layers of protection dedicated for preserving fidelity during translation of alanine codons^24,25. All the AlaRS-Ed members bind a Zn²⁺ ion through universally conserved motif: HXXXH and CXGXH^26,27,28,29. ThrRS in bacteria, eukarya and some archaeal members possess a cis-editing domain called as N2 domain for correcting the mischarged product Ser-tRNA^Thr (From here onwards N2 domain will be referred to as ThrRS-Ed).

The AlaRS-Ed and ThrRS-Ed domains are homologous²¹ and structurally both belong to ThrRS/AlaRS editing domain-like superfamily (SCOP ID: 3001504). Apart from structural and sequence homology, both AlaRS-Ed and ThrRS-Ed exhibit a striking resemblance in their catalytic sites featuring identical Zn²⁺-binding motif HXXXH and CXGXH²¹ (Supplementary Fig. 2a, b). Mutational analysis established that Cys in CXGXH motif in both the editing domains is crucial for proofreading activity^22,30,31,32. Moreover, studies have shown that the proofreading activity of E. coli ThrRS (EcThrRS) is affected by reactive oxygen species (ROS) metabolite, hydrogen peroxide (H₂O₂), through the oxidative modification of thiol (-SH) R-group of C182 (CXGXH)^33,34. However, the proofreading activity of E. coli AlaRS (EcAlaRS) on the other hand is resistant to the ROS (H₂O₂)³⁵. Furthermore, experiments in E. coli showed that mistranslation of Thr-codons arising from defect in proofreading activity of ThrRS is well tolerated by the cells, whereas, defect in AlaRS proofreading activity causes growth defect in E. coli and S. cerevisiae^22,36,37. Remarkably, even a mild compromise in AlaRS proofreading activity in mouse leads to severe neurological and cardiac disorders^38,39. The disparity in cellular tolerance for the mischarging by AlaRS vs. ThrRS suggests that the distinct sensitivity of their proofreading activity towards oxidative stress possibly caters to the cellular needs for differential mistranslation of Ala- vs. Thr-codons.

The molecular underpinnings governing the dichotomy in sensitivity of the activities of AlaRS-Ed and ThrRS-Ed toward ROS, despite featuring a strikingly homologous structure and identical Zn²⁺ binding motif remains unknown. Here, we reveal that the Zn²⁺ ion bound to AlaRS-Ed is dispensable for both structural integrity as well as proofreading activity. Based on in vitro and in vivo results, we show that the metal ion bound to AlaRS-Ed protects its biochemical activity to maintain accuracy of Ala-codons translation under oxidative stress. Whereas, the ThrRS-Ed allows facile abrogation of its proofreading activity by ROS, thereby enabling adaptive mistranslation of Thr-codons. The study further demonstrates how a subtle alteration in the design of metal binding sites leads to differential ROS vulnerability and gave rise to one of the earliest regulatory framework that links redox metabolism with translation quality control.

Results

Universally conserved Zn²⁺ ion in AlaRS-Ed is dispensable for both structure and proofreading activity

Editing domain of AlaRS is universally conserved and features invariant HXXXH and CXGXH Zn²⁺ -binding motifs that coordinate a Zn²⁺ ion (Supplementary Fig. 1a, b). Previous studies have indicated that Zn²⁺ bound to AlaRS-Ed may be dispensable for enzymatic activity on mischarged product Ser-tRNA^Ala ^27,29. However, the involvement of Zn²⁺ in cognate discrimination and structural stability was not determined, thereby leaving open the question of what is the necessity to preserve a highly conserved Zn²⁺ binding site across all domains of life? To examine the role of Zn²⁺ in AlaRS-Ed, we removed the bound Zn²⁺ from purified EcAlaRS WT and a trans-editing factor from an archaeon Pyrococcus horikoshii, PhoAlaX-M, by chelating with a strong Zn²⁺ chelator 1,10-phenanthroline²⁶ and confirmed by colorimetric assay using 4-(2-Pyridylazo) resorcinol (PAR) dye (Supplementary Fig. 3a, b)⁴⁰. Next, we deacylated Ala-(Ec)tRNA^Ala, Ser-(Ec)tRNA^Ala and Gly-(Ec)tRNA^Ala by holo- and apo-form of EcAlaRS and PhoAlaX-M. Deacylation assays revealed that both holo- and apo-form of EcAlaRS and PhoAlaX-M enzymes deacylate Ser- and Gly-(Ec)tRNA^Ala with equal efficiency, and both were inactive on cognate Ala-(Ec)tRNA^Ala (Fig. 1a–c and Supplementary Fig. 3e–j). Thus, in evolutionarily distant homologues of AlaRS-Ed, Zn²⁺ ion is not required for enzymatic activity and substrate discrimination, thereby, establishing the generality of the dispensability of Zn²⁺ for proofreading activity of the AlaRS-Ed family.

**Fig. 1: Zn²⁺ in AlaRS-Ed is dispensable for structure and proofreading activity.**

Zn²⁺ ion is present in multiple AlaRS-Ed proteins when purified from the cells (Supplementary Fig. 3a, b) and multiple atomic structures of AlaRS-Ed deposited in PDB database with resolution of 2.6 Å or better, including the PfuAlaX-M structure solved in this study (Supplementary Table 1 and Supplementary Fig. 3l), have Zn²⁺ ion coordinated via HXXXH and CXGXH motifs (Fig. 1d, e and Supplementary Figs. 1a, b and 3k). However, in two of the crystal structures deposited in PDB database with resolution of 2.6 Å or better, PhoAlaX-S (PDB: 1V7O) and PhoAlaRS (PDB: 2ZZE) do not have Zn²⁺ ion bound to HXXXH and CXGXH motif. Structures of PhoAlaX-S apo and PhoAlaRS cis-Ed apo superimpose on AfuAlaRS cis-Ed holo with r.m.s.d. of ~1.3 Å over 135 Cα atoms and ~1.0 Å over 235 Cα atoms respectively, without any discernible structural perturbation arising from presence or absence of Zn²⁺ (Fig. 1f and Supplementary Fig. 3m). Further, we performed protein thermal shift assay with holo- and apo-form of EcAlaRS and PhoAlaX-M proteins to examine the effect of Zn²⁺ on thermal stability of AlaRS-Ed. Zn²⁺ coordination residue mutant proteins, H568A in EcAlaRS and H103A in PhoAlaX-M, that lacks the ability to bind Zn²⁺ were also included in the assay (Supplementary Fig. 3c, d). Melting temperature (Tm) of EcAlaRS WT-holo is 54.7 °C and the Tm of both WT-apo and H568A are ~1 °C less than that of WT-holo, showing that removal of Zn²⁺ does not have any notable effect on thermal stability of EcAlaRS protein (Fig. 1g, i). Similarly, Tm of PhoAlaX-M WT-holo is 92.5 °C and the Tm of both WT-apo and H103A mutant are ~1–2 °C less than that of WT-holo, showing that Zn²⁺ ion does not play notable role in thermal stability of PhoAlaX-M as well (Fig. 1h, i). Altogether, the in vitro activity and thermal stability assay results coupled with insights gleaned through structural analysis presented here establish that the universally conserved Zn²⁺ ion bound to AlaRS-Ed is dispensable for both structural integrity as well as proofreading activity.

Distinct Zn²⁺ binding underpins opposing effect of ROS on activity of AlaRS-Ed and ThrRS-Ed

AlaRS-Ed and ThrRS-Ed belong to the ThrRS/AlaRS editing domain-like superfamily, and additionally, both feature identical HXXXH and CXGXH Zn²⁺-binding motif (Fig. 2a, b and Supplementary Fig. 2a, b)²¹. Zn²⁺ is always present bound to the active AlaRS-Ed proteins purified from the cells, whereas in the case of ThrRS-Ed protein purified from E. coli, Zn²⁺ is not detectable by PAR assay (Supplementary Figs. 3a, b and 4a). Next, we performed PAR-based competition assay to estimate the Zn²⁺ binding affinity of AlaRS-Ed and ThrRS-Ed^41,42. Competition assay revealed that dissociation constant for Zn²⁺ binding by PhoAlaX-M WT is ~0.8 ± 0.2 nM, which is ~80-fold less than EcThrRS-Ed (only the editing domain region of ThrRS comprising of N-terminal domains N1 and N2; Supplementary Fig. 2a) that has dissociation constant for Zn²⁺ binding of ~64 ± 11.13 nM (Fig. 2c and Supplementary Fig. 4b). Thus, AlaRS-Ed has more affinity for Zn²⁺ than ThrRS-Ed. Moreover, in a previous study, it was mentioned that Zn²⁺ inhibit ThrRS-Ed activity but the data regarding the same was not presented³⁰. To understand the effect of free Zn²⁺ ion on the activity of homologous AlaRS-Ed and ThrRS-Ed, we checked their deacylation activities on Ser-(Ec)tRNA^Ala and Ser-(Ec)tRNA^Thr respectively, in the presence and absence of free Zn²⁺ in the reaction mixture. Deacylation assays revealed that ≥50 µM free Zn²⁺ concentration in the reaction inhibits EcThrRS WT activity, whereas even 1000-fold higher free Zn²⁺ concentration in the reaction did not affect the deacylation activity of EcAlaRS WT and PhoAlaX-M WT (Fig. 2d, e and Supplementary Fig. 4c). Thus, free Zn²⁺ has opposing effect on the proofreading activity of the homologous AlaRS-Ed and ThrRS-Ed domains.

**Fig. 2: Opposing effect of Zn²⁺ and ROS on activity of homologous AlaRS-Ed and ThrRS-Ed.**

Intriguingly, it has been shown that the activity of EcThrRS-Ed is affected by H₂O₂^33,34, a constituent of cellular redox metabolism⁴³, whereas the activity of homologous EcAlaRS-Ed is impervious to the same³⁵. In case of ThrRS-Ed, oxidation of C182, which is involved in substrate binding through A76 2’-OH of Ser-tRNA^Thr ³⁰, results in disruption of substrate binding and thereby abrogation of activity³⁴. Moreover, Zn²⁺ is not involved in catalysis in ThrRS-Ed, rather its presence is inhibitory for the activity due to preclusion of substrate binding residue C182 and rearrangement of catalytically important H73 (Fig. 2f, g). Furthermore, AlaRS-Ed and ThrRS-Ed are homologous and share the same Zn²⁺ binding motif that harbour the catalytically important cysteine and also the substrates they act on are similar, implying that they share a common catalytic mechanism. Even after sharing the aforementioned commonalities, ThrRS-Ed’s activity is affected by ROS due to oxidation of the substrate binding cysteine, whereas, the activity of AlaRS-Ed is resistant to ROS despite featuring the homologous catalytically important cysteine. The observed distinction between AlaRS-Ed and ThrRS-Ed with regard to Zn²⁺-binding prompted us to hypothesise that the universally conserved Zn²⁺ ion bound to catalytically important cysteine in AlaRS-Ed may prevent the cysteine’s oxidation and thereby confers protection to its activity from ROS. We employed in vitro deacylation assay to biochemically examine the effect of H₂O₂ on the proofreading activity of holo- and apo-form of EcAlaRS and PhoAlaX-M (Supplementary Fig. 4d). Deacylation assays revealed that, upon treatment with H₂O₂, the proofreading activity of EcAlaRS-holo and PhoAlaX-M-holo were unperturbed, however, EcAlaRS-apo and PhoAlaX-M-apo enzymes showed pronounced defect in deacylation of Ser-(Ec)tRNA^Ala (Fig. 2h, i and Supplementary Fig. 4e, f). The in vitro deacylation assay results pertaining to both AlaRS and AlaX-M establish that the universally conserved Zn²⁺ in AlaRS-Ed plays a protective role for its activity against reactive ROS-metabolites like H₂O₂.

Next, we sought to probe the modification of Cys (CXGXH) in AlaRS-Ed holo- and apo-form upon treatment with H₂O₂. A previous work reported that EcAlaRS WT upon treatment with H₂O₂, where Zn²⁺ was not removed, leads to sulfenic acid modification of C666 albeit without any effect on the enzymatic activity³⁵. This observation is puzzling as to how despite sulfenic acid modification of C666, EcAlaRS-Ed still preserves its activity, in contrast to what is observed in the case of EcThrRS-Ed^33,34. Importantly, in another study multiple aaRSs, including ThrRS, were shown to contain sulfenic acid modification in HeLa cells, whereas such a modification was not detected in AlaRS⁴⁴. Hence, we undertook an alternative approach to probe the modification of Cys residue upon treatment with H₂O₂ using Ellman’s reagent (DTNB). Sulfhydryl group in Cys residues reacts with DTNB to produce yellowish coloured TNB^2-, which has an absorbance maximum at λ_max = 412 nm⁴⁵, however, oxidised Cys residues do not react with DTNB and therefore become undetectable in the same assay (Fig. 2j)³³. We used PhoAlaX-M WT protein for the assay as it contains a single Cys residue as part of the CXGXH Zn²⁺ binding motif. PhoAlaX-M WT holo, PhoAlaX-M WT apo, PhoAlaX-M C182A and EcN1 + N2 WT apo were treated with H₂O₂ and then assayed for the presence of sulfhydryl group using Ellman’s reagent. The level of sulfhydryl group in PhoAlaX-M WT holo remain unchanged upon treatment with H₂O₂, whereas the level of sulfhydryl group depleted to no protein control level in case of PhoAlaX-M WT apo and ThrRS-Ed proteins (Fig. 2k). No sulfhydryl group was detectable in PhoAlaX-M C182A mutant protein. The Ellman’s reagent assay results clearly show that the sulfhydryl group of catalytically important Cys residue in AlaRS-Ed, which is bound to Zn²⁺, is protected from oxidation by H₂O₂, whereas the corresponding Cys in homologous ThrRS-Ed is readily oxidised by the same. Overall, the universally conserved Zn²⁺ bound to AlaRS-Ed protects its activity from ROS by shielding a catalytically important Cys residue from getting oxidised.

Engineering an AlaRS-Ed variant customised for probing the role of its bound Zn²⁺ in vivo

In order to examine the protective effect of the Zn²⁺ bound to AlaRS-Ed from ROS in the cellular context, we sought to design an AlaRS-Ed variant that retains all the native functional characteristics except for binding the Zn²⁺ ion—akin to ThrRS-Ed. A previous study has reported that mutation of a conserved H568 in ⁵⁶⁴HXXXH⁵⁶⁸/⁶⁶⁶CXXXH⁶⁷⁰ motifs in EcAlaRS that coordinate the Zn²⁺ ion does not affect the editing activity (Fig. 3a)²². Indeed, the EcAlaRS H568A and PhoAlaX-M H103A mutant proteins do not bind Zn²⁺ when purified from cells (Supplementary Fig. 3c, d) and competition assay with PAR revealed PhoAlaX-M H103A has reduced affinity for Zn²⁺ binding (Kd of ~20 ± 2.8 nM) (Supplementary Fig. 5a, b). We then checked the deacylation activity of both proteins on Ser- and Gly-(Ec)tRNA^Ala. The deacylation assay showed that both the proteins could efficiently deacylate Ser- and Gly-(Ec)tRNA^Ala. Next, we pretreated EcAlaRS H568A and PhoAlaX-M H103A enzymes with H₂O₂ before checking their deacylation activity. The activity of both the enzymes gets affected upon treatment with H₂O₂ (Fig. 3b and Supplementary Fig. 5c, e). Further, to ascertain that the His (HXXXH) mutant enzymes retain the substrate discrimination capability and avoid cognate-misediting, we checked their activity on Ala-(Ec)tRNA^Ala. Both the mutant enzymes were inactive on Ala-(Ec)tRNA^Ala (Fig. 3c and Supplementary Fig. 5d), thereby confirming that the cognate discrimination capability of the mutant has not been perturbed. Furthermore, we titrated the EcAlaRS H568A enzyme with decreasing concentration of H₂O₂ (1 mM, 0.1 mM and 0.05 mM) and then checked the effect on deacylation activity. We observed that deacylation activity of EcAlaRS H568A is affected by as low as 0.05 mM H₂O₂ and the effect gets pronounced with increasing H₂O₂ concentration (Fig. 3b). The 50 µM concentration of H₂O₂ where we begin to see the effect on editing activity of EcAlaRS H568A is a physiologically relevant H₂O₂ concentration^33,46,47. Ellman’s reagent assay with PhoAlaX-M H103A revealed that the sulfhydryl group of catalytically important C182 gets readily oxidised upon treatment of H₂O₂ (Fig. 3d). Overall, the EcAlaRS H568A mutant, owing to its higher sensitivity to H₂O_2, without requiring removal of Zn²⁺ by a chelator, behaves in a similar manner to ThrRS-Ed. Thus, the above mutant is suitable for in vivo studies to interrogate the role of Zn²⁺ towards protection of AlaRS-Ed from ROS in the cellular context.

**Fig. 3: Zn²⁺ protects AlaRS editing activity from ROS in vivo.**

Zn²⁺ protects AlaRS-Ed activity from ROS and prevents Ala-codon mistranslation in vivo

We set out to check whether the protection of AlaRS proofreading activity from ROS by Zn²⁺ is relevant in physiological scenario inside cells. For this purpose, we knocked out alaS gene in Escherichia coli MG1655 native strain by phage-mediated method with a shelter plasmid containing either ^EcalaS^WT or the ^EcalaS^H568A mutant gene. Since DTD is a redundant Gly-tRNA^Ala proofreader, we used Δdtd strain generated previously by Pawar et al.⁴⁸ to make the double knock-out strains MG1655ΔalaS Δdtd (DKO), complemented with either ^EcalaS^WT or ^EcalaS^H568A. Next, we performed spot-dilution based cell viability assay to investigate the effect of oxidative stress on their fitness (Fig. 3e). Overall, both the strains harbouring either ^EcalaS^WT or ^EcalaS^H568A had comparable growth in control condition. In presence of 3 mM serine or 10 mM glycine, the strain harbouring ^EcalaS^WT grew normally, but the strain harbouring ^EcalaS^H568A had notable growth defect with serine and marginal defect with glycine compared to the control. Remarkably, addition of H₂O₂ on top of non-cognate amino acids to the growth media completely abolished the growth of strain harbouring ^EcalaS^H568A, but the strain harbouring ^EcalaS^WT did not show any notable growth defect. Furthermore, to ascertain whether the observed toxicity is indeed due to misaminoacylation by AlaRS, 10 mM alanine was supplemented in the growth media on top of non-cognate amino acids and H₂O₂. Indeed, the supplementation of alanine in the growth media substantially alleviated the growth defect of the strain harbouring ^EcalaS^H568A caused by non-cognate amino acids and H₂O₂. Thus, Zn²⁺ ion bound to AlaRS-Ed protects its activity from ROS in the cells.

Next, we sought to investigate whether the conditional fitness defect seen in E. coli strain harbouring AlaRS-Ed that lacks Zn²⁺ is due to mistranslation of Ala-codons in the cells. We employed a GFP-based cellular reporter to probe the Ala>Gly mistranslation in E. coli⁴⁹. In this reporter, the G67 in the ⁶⁵TYG⁵⁰ tri-peptide critical for chromophore formation in super folder GFP (sfGFP) is substituted to alanine, sfGFP G67A, which renders it non-fluorescent (Fig. 3f). Ala-codons mistranslation in a cell would result in synthesis of sfGFP with ⁶⁵TYG⁵⁰, thereby recover GFP-fluorescence signal non-genetically in sfGFP G67A background. We introduced gfp^G67A in DKO strains complemented with either ^EcalaS^WT or ^EcalaS^H568A. These strains were then grown in media supplemented with or without 50 µM H₂O₂ and 10 mM glycine. Microscopic examination of the strains revealed that strain harbouring AlaRS H568A exhibits GFP-fluorescence upon growth in media containing H₂O₂ and glycine but not in control condition, whereas strain harbouring AlaRS WT did not show any fluorescence in both the conditions (Fig. 3g). The recovery of GFP-fluorescence in bacterial cells with sfGFP G67A shows occurrence of Ala-codon mistranslation in strain harbouring AlaRS H568A but not in the strain harbouring AlaRS WT. Thus, our observations from cell viability and cellular mistranslation reporter assays clearly establish that Zn²⁺ ion bound to AlaRS-Ed shields its proofreading activity from ROS and prevent the toxic mistranslation of Ala-codons in cells.

Structural basis of distinct Zn²⁺ binding by homologous AlaRS-Ed and ThrRS-Ed

Despite featuring the same fold and identical Zn²⁺-binding motifs, AlaRS-Ed binds a Zn²⁺ ion that protects its proofreading activity from ROS, whereas in case of ThrRS-Ed Zn²⁺ binding is undetectable by PAR assay and its activity is readily affected by ROS. To understand the molecular basis underlying the difference in Zn²⁺ binding between AlaRS-Ed and ThrRS-Ed, we analysed the structures of both proteins available in PDB database with resolution of 2.5 Å or better, including the structure of PfuAlaX-M solved in this study (Fig. 1d, e and Supplementary Fig. 6a) (Supplementary Table 1 and Supplementary Fig. 3l). A structural comparison was done by superimposing AfuAlaRS-Ed (PDB: 2ZTG)⁵¹ and EcThrRS-Ed (PDB: 1TKE)³⁰, with an r.m.s.d. of ~2.4 Å over 135 Cα atoms (Fig. 4a). The structural comparison revealed that in AlaRS-Ed the ligand residues are sterically well organised to form a tetrahedral coordination sphere for binding a Zn²⁺ ion, whereas they are distorted in ThrRS-Ed (Fig. 4b). To compare the coordination spheres, we measured the distance between ligand residues in AlaRS-Ed and ThrRS-Ed structures. The inter-ligand distance analysis revealed that the coordination sphere in ThrRS-Ed is notably larger than AlaRS-Ed’s due to distinct positioning of ligand residues (Fig. 4b and Supplementary Fig. 6a,). The loop region spanning CXGXH motif in AlaRS-Ed and ThrRS-Ed structures have different orientation in all the analysed structures resulting in distinct positioning of the Zn²⁺ ligand Cys in both the structures (Fig. 4b, d). Upon a close inspection, we found that an invariant Y173, a part of hydrophobic shell residues surrounding the coordination sphere, in the case of ThrRS-Ed pushes the loop harbouring C182 (CXGXH) ~ 2.4 ± 0.2 Å away relative to the loop’s position in AlaRS-Ed (Supplementary Fig. 6b). Strikingly, only non-bulkier residues V/I/L/T occupy the position in AlaRS-Ed corresponding to Y173 of ThrRS-Ed (Fig. 4b, c and Supplementary Fig. 7a). Structural superimposition of the two structures reveal that the side chain of the conserved Y173 in ThrRS-Ed encounters steric clash with residues in the CXGXH loop in AlaRS-Ed that is positioned appropriately to form a coordination sphere competent for Zn²⁺ binding (Fig. 4b and Supplementary Fig. 7b–e). Notably, the side chain –OH group of Y173 in all the ThrRS-Ed structures encounter steric clash (average distance 1.7 ± 0.3 Å) with the main chain carbonyl group of G705 (CXGXH) in all the AlaRS-Ed structures (Fig. 4b). In case of ThrRS-Ed the corresponding residue G184’s (CXGXH) main-chain carbonyl oxygen has flipped ~180° away relative to G705 in AlaRS-Ed, averting any steric clash (Fig. 4b, e and Supplementary Fig. 7c, d). The distinct orientation of glycine in CXGXH loop of AlaRS-Ed vs. ThrRS-Ed entails switch in Ramachandran angles by ~150° of Phi (Ф) and ~70° of Psi (Ψ) angles (Fig. 4f–h) and notably no other residue in CXGXH motif shows any significant difference in Ramachandran angles between the two structures (Supplementary Fig. 8f). The flip in Ramachandran angles of central glycine alters the conformation of loop spanning CXGXH motif in AlaRS-Ed vs. ThrRS-Ed resulting in distinct steric organisation of the coordination spheres that determines distinct Zn²⁺ binding by the respective proteins.

**Fig. 4: Structures reveal determinant of difference in Zn²⁺ binding by AlaRS-Ed and ThrRS-Ed.**

Next, we analysed the ThrRS structure from Staphylococcus aureus (SaThrRS) (PDB: 1NYR)⁵² where one of the protomers has a Zn²⁺ ion bound to its editing domain (SaThrRS-Ed holo) (Supplementary Fig. 9a–d). Strikingly, the side-chain of conserved Y172, a part of hydrophobic shell in SaThrRS-Ed holo, has rotated ~130° away relative to Y173 in ThrRS-Ed apo (Supplementary Fig. 9e). The G183 (CXGXH) in SaThrRS-Ed holo orients similar to AlaRS-Ed albeit without any clash with Y172 (Fig. 4e). The reorientation of G183 in SaThrRS-Ed holo entails changes in its Ramachandran angles and concomitantly the loop spanning CXGXH motif assumes a path partially similar to AlaRS-Ed ensuing formation of a coordination sphere competent for binding Zn²⁺ (Fig. 4f–h). Thus, analysis of ThrRS-Ed apo and ThrRS-Ed holo structures illuminate how the invariant hydrophobic shell residue Y173 in ThrRS-Ed renders the coordination sphere incompetent for binding Zn²⁺, which can be partially reversed by a ~130° rotation of its side chain. Overall, the structural analysis reveal that, despite featuring homologous structure and identical Zn²⁺-binding motifs, AlaRS-Ed and ThrRS-Ed have diverged in Zn²⁺ binding by virtue of a difference in a key hydrophobic shell residue surrounding their coordination spheres.

A single residue switch renders AlaRS-Ed ROS sensitive

In order to functionally test the insights gleaned through structural analysis, we generated mutant proteins: L655Y in EcAlaRS, T172Y in PhoAlaX-M and Y173V in EcThrRS-Ed (N1 + N2). EcAlaRS L655Y binds Zn²⁺ albeit with a decrease in affinity, as the Zn²⁺ binding dye PAR can sequester Zn²⁺ from EcAlaRS L655Y to more extent than EcAlaRS WT (Fig. 5a, b). PAR assay and PAR-based competition assay revealed that PhoAlaX-M T172Y has reduced Zn²⁺ binding affinity (Supplementary Fig. 10a–c). However, the reverse mutation EcThrRS-Ed Y173V did not show any trace of gain-of-function in Zn²⁺ binding compared to EcThrRS-Ed WT, suggesting the presence of other epistatic anti-determinant of Zn²⁺-binding (Fig. 5c). Next, we performed in vitro deacylation assay to check the effect of H₂O₂ on the activity of EcAlaRS WT, EcAlaRS L655Y, PhoAlaX-M WT, PhoAlaX-M T172Y on Ser-(Ec)tRNA^Ala, Gly-(Ec)tRNA^Ala and Ala-(Ec)tRNA^Ala. The deacylation assay showed that EcAlaRS L655Y enzyme could efficiently deacylate Ser-(Ec)tRNA^Ala and Gly-(Ec)tRNA^Ala similar to EcAlaRS WT (Fig. 5d, e and Supplementary Fig. 10d). However, upon treatment with H₂O₂ the activity of EcAlaRS L655Y on both Ser-(Ec)tRNA^Ala and Gly-(Ec)tRNA^Ala gets severely affected, whereas activity of EcAlaRS WT remain unaffected by the same (Fig. 5d, e and Supplementary Fig. 10d). Similarly, the activity of PhoAlaX-M T172Y on Ser-(Ec)tRNA^Ala is also affected by 5 mM H₂O₂ (Supplemental Fig. 10e). Moreover, both EcAlaRS L655Y and PhoAlaX-M T172Y did not have any activity on cognate Ala-(Ec)tRNA^Ala, implying that the mutation does not affect substrate discrimination property of the enzymes (Supplementary Fig. 10f, g). Ellman’s reagent assay with PhoAlaX-M T172Y revealed that the sulfhydryl group of catalytically important C182 gets readily oxidised upon treatment with H₂O₂ (Fig. 5f).

**Fig. 5: Implanting ThrRS-Ed determinant in AlaRS-Ed affects its Zn²⁺ binding and ROS resistance of activity.**

Furthermore, we sought to test the biochemical observation that L/T > Y mutation in AlaRS-Ed renders it sensitive to ROS, in cellular context. We employed spot dilution-based viability assay to examine the fitness of E. coli MG1655 DKO strain complemented with either ^EcalaS^WT or ^EcalaS^L655Y mutant genes under oxidative stress (Fig. 5g). Both the strains grew normally in control condition and upon supplementation of 3 mM serine. The strain harbouring ^EcalaS^L656Y showed notable growth defect in presence of H₂O₂ compared to the strain harbouring ^EcalaS^WT. Moreover, presence of both H₂O₂ and serine together in media completely abolished the growth of strain harbouring ^EcalaS^L656Y, compared to the strain with ^EcalaS^WT. The growth defect observed in ^EcalaS^L656Y strain in presence of H₂O₂ and serine is completely rescued by supplementing cognate amino acid alanine, implying that the observed growth defect is indeed rooted in AlaRS mischarging. Overall, both the in vitro and in vivo experimental evidences are consistent with the insights gleaned through structural analysis, which together establish that AlaRS-Ed and ThrRS-Ed have diverged in Zn²⁺ binding by virtue of a switch in hydrophobic shell residue surrounding the coordination sphere.

Pre-LUCA origin of the functional dichotomy between AlaRS-Ed and ThrRS-Ed

To gain insights into the evolutionary basis of the divergence between AlaRS-Ed and ThrRS-Ed, we undertook a phylogenetic analysis based approach to infer the evolutionary relationship between the extant AlaRS-Ed and ThrRS-Ed sequences. We reconstructed Maximum likelihood (ML) phylogenetic tree of AlaRS cis-Ed, AlaXps and ThrRS-Ed sequences from organisms belonging to archaea, bacteria and eukarya. In the ML phylogenetic tree, all the ThrRS-Ed sequences from all the three domains of life show a monophyletic distribution, indicating that the divergence between AlaRS-Ed and ThrRS-Ed happened before two of the domains of life—archaea and bacteria—diverged at the stage of LUCA (Fig. 6a). However, ThrRS-Ed (N2-domain) is not present universally, but in ~30% of archaeal genomes, whereas the rest of the archaeal genomes have an analogous NTD-type ThrRS editing domain. The non-universal distribution of ThrRS-Ed in archaeal branch raises an alternative possibility that ThrRS-Ed was introduced in archaeal organisms by horizontal gene transfer, and therefore may not have been present in LUCA. In order to ascertain whether ThrRS-Ed was present in LUCA, we set out to infer the phylogenetic relationship between ThrRS-Ed sequences in archaea and bacteria. We reconstructed separate ML phylogenetic trees, one for N1 + N2 domains and other for the rest of the universally conserved region of ThrRS spanning aminoacylation and anticodon binding domain (AD + ABD) (Supplementary Fig. 2a), using archaeal and bacterial sequences. Both the phylogenetic trees corresponding to N1 + N2 and AD + ABD regions of ThrRS feature similar topology and exhibit domain separation of bacterial and archaeal sequences, implying their presence in LUCA (Supplementary Fig. 11a,b). We also reconstructed ML phylogenetic tree of AlaRS, a bona fide LUCA protein, using sequences belonging to same set of organisms employed in ThrRS phylogenetic trees (Supplementary Fig. 11c). The phylogenetic tree of AlaRS also features a topology similar to N1 + N2 and AD + ABD trees, exhibiting domain separation of bacterial and archaeal sequences as expected for a protein present in LUCA. Importantly, geological and genomic studies suggest that LUCA encountered abiotically generated H₂O₂ and it already possessed oxidative stress defence related genes^{53,54,55,56,57}. Thus, our extensive phylogenetic analysis implies the existence of ThrRS-Ed in LUCA and thereby favours a scenario of pre-LUCA divergence between AlaRS-Ed and ThrRS-Ed driven by ROS (Fig. 6b).

**Fig. 6: Ancient divergence between ThrRS-Ed and AlaRS-Ed.**

Discussion

The ThrRS/AlaRS editing domain-like superfamily represents a major class of proofreading domain in class II aaRSs that shows universal distribution and present from LUCA, which is paralleled by only CP1 editing domain found in class I aaRSs². Despite the vast body of work carried out on this superfamily, over the past several decades, it remained unclear as to what role the metal binding site plays that underlie its universal conservation through ~3.5 billion years of evolution.

Here, we identify the hitherto unknown function of the Zn²⁺ ion bound to AlaRS-Ed, which is dispensable for both structure as well as proofreading function, for protection of its activity from ROS and prevent toxic mistranslation of Ala-codons in cells. The conditional lethality seen in E. coli harbouring AlaRS-Ed faulty in Zn²⁺ binding highlights the importance of this cellular function under oxidative stress. The protection offered to AlaRS-Ed during oxidative stress thus provides a rationale as to why Zn²⁺ binding in this domain is universally conserved. The challenging substrate discrimination AlaRS-Ed has to achieve⁵⁸ compounded by the requirement for protection of a catalytic residue from cellular ROS imposes stringent constrains that a unique solution evolved only once and remains conserved through ~3.5 billion years of evolution since LUCA. It is worth noting here that such a solution is also adopted in all the redundant trans-editing variants of AlaRS-Ed that act as multiple sieves dedicated for solving a critical problem in translation quality control.

In stark contrast, increased tolerance for Thr-to-Ser mistranslation allowed speciation of a ROS-switchable proofreading function of homologous ThrRS-Ed, also at the level of LUCA itself^9,12. Thus, a Zn²⁺ ion mediated functional dichotomy of homologous AlaRS-Ed and ThrRS-Ed encodes mistranslation landscape of Ala- vs Thr-codons in response to oxidative stress, moulded by the disparity in penalty associated with mistranslation of respective amino acids. It is appealing to propose here that the potential for adaptive modulation of ThrRS-Ed activity could be the underlying selective advantage for its recruitment in eukaryotes over the NTD-type ThrRS editing domain more prevalent in archaea^59,60,61,62, whose structural homologue Animalia-specific-tRNA deacylase’s (ATD) activity has been shown to be resistant to oxidative stress⁶³.

The inhibition of ThrRS-Ed proofreading activity by Zn²⁺ was noted earlier, even though the data was not shown³⁰ and is corroborated in the current study. It is well established that oxidative stress gives rise to spike in cellular flux of free Zn²⁺ that modulates function of various proteins^64,65. Thus, oxidative stress potentially abrogates proofreading activity of ThrRS-Ed via two different mechanisms in concert—modification of catalytic site residue and Zn²⁺ binding—an aspect that warrants further investigation. On the contrary, AlaRS-Ed always binds a Zn²⁺ ion and its activity is unaffected by even high concentrations of free Zn²⁺ in the reaction mixture. Besides, the striking commonalities between AlaRS-Ed and ThrRS-Ed with regard to their structure, catalytic site residues, reaction they catalyse and the substrates they act upon, strongly implicate a common mechanism of catalysis shared by both the enzymes. Moreover, we show that Zn²⁺ is dispensable for proofreading activity as well as structural integrity of AlaRS-Ed, and an analysis of the available structures indicates that mechanistically the inhibitory nature of Zn²⁺ in ThrRS-Ed catalytic site could be mirrored by AlaRS-Ed as well (Fig. 2g).

In an attempt to reconcile the observed distinction between AlaRS-Ed and ThrRS-Ed regarding the effect of Zn²⁺ on their respective activities vis-à-vis a common mode of catalysis, we hypothesise and propose a ‘migratory’ Zn²⁺ mechanism where it transits in AlaRS-Ed to a secondary site (2°) in presence of substrate and post-catalysis returns to the primary site (1°) formed by HXXXH/CXGXH motifs upon release of product (Fig. 6b). In this context, it is worth noting here that the observed position occupied by Zn²⁺ in various AlaRS-Ed structures display a mobile nature (Supplementary Fig. 12a). It implies that the Zn²⁺ ion bound to AlaRS-Ed is not absolutely rigid and exhibit a certain degree of freedom albeit in a manner not to allow the catalytic cysteine oxidation in between reaction cycles. This aspect is consistent with the hypothesised migration of Zn²⁺ ion out of its canonical position during catalysis. In this regard, solution of the complex structure with substrate mimicking analogue bound to the catalytic site may enable unravelling such an unprecedented catalytic mechanism of AlaRS-Ed.

Redox metabolism is inherently linked to the energy metabolism of a cell, and therefore, plays a fundamental role in cellular homoeostasis by enabling network of interactions connecting cellular energy state with myriad of other processes^43,66,67. The unique chemistry of thiol R-group of cysteine residues in proteins endow them with enzymatic activity, redox reactivity and metal binding capabilities, a confluence of which allows these proteins to integrate cellular redox state and free Zn²⁺ flux into their functionality^{50,65,68,69,70,71}. Many protein families like phosphatases, kinases, proteases, Zn-finger containing transcription factors, etc., are influenced by redox-signalling owing to the ability of their members to interact with cellular flux of ROS metabolites and/or free Zn²⁺ ^50,65,72. The non-degeneracy noted in the effect of ROS and/or Zn²⁺ on activity could represent an organisational principle for encoding the landscape of redox metabolism based regulatory networks that control and coordinate important cellular processes^{67,73,74,75,76}.

In this study we show how speciation in metal binding gave rise to distinct sensitivity of activity towards ROS in members of the same superfamily of enzymes, which encodes mistranslation landscape of Ala- vs Thr-codons. Thus, the distinct metal binding driven functional dichotomy plays a key role in organisation of one of the most ancient regulatory network that integrates redox signalling and translation quality control (Fig. 6c). While such coupling has been seen in extant organisms including pathogenic bacteria and higher eukaryotic systems, it is striking to note that such crucial linkages are rooted in LUCA and this study has identified one such unique example of possibly many more that remains to be explored. It is also worth underscoring in this context that molecules that are central to the origin and evolution of the genetic code are involved at a very early stage for stress dependent alteration of the proteome.

Methods

Sequence and structural analysis

All the sequences of AlaRS, AlaXps and ThrRS were retrieved from KEGG GENES database using KEGG BLAST search (https://www.genome.jp/tools/blast/). Structure-based multiple sequence alignment of the sequences was performed using Expresso programme in T-coffee server⁷⁷. Alignments were curated in Jalview⁷⁸. The Maximum likelihood phylogenetic trees were reconstructed from the structure-based multiple sequence alignment using online programme IQ-tree (http://iqtree.cibiv.univie.ac.at/)⁷⁹. The phylogenetic trees were visualised in iTol⁸⁰.

Atomic coordinates of the structures were downloaded from RCSB protein data bank. Structural superimposition of the structures was carried out in Coot⁸¹ using SSM method. For superimposition of AlaRS-Ed and ThrRS-Ed structures only the N2 region of both the proteins were generated in Pymol and then superimposed in Coot using SSM method. Inter-atomic clash in the structures was probed in Pymol using Protein Interaction Viewer plugin⁸².

Cloning, expression and purification of proteins

Escherichia coli AlaRS N-terminal 6X histidine tagged (EcAlaRS WT N-His) was cloned, expressed and purified as described earlier⁴⁸. AlaX-M gene from Pyrococcus furiosus (Pfu) and Pyrococcus horikoshii (Pho) were PCR-amplified from respective genomic DNA (received from ATCC) and cloned into pET28b expression vector (Novagen). E. coli ThrRS (EcThrRS) gene was PCR-amplified from genomic DNA and cloned into pET28b vector with N-terminal 6X histidine tagged (N-His) using restriction free cloning method. EcN1 + N2 without tag was generated by PCR-amplified from genomic DNA and cloned into pET28b vector using restriction free cloning. Site-directed mutagenesis were done to generate mutant proteins using DNA oligos listed in Supplementary Table 2.

The recombinant proteins were overexpressed in E. coli BL21 (DE3) host expression strains and purified through two-step method comprising of affinity chromatography followed by size exclusion chromatography (SEC). Untagged PfuAlaX-M WT, PhoAlaX-M WT, PhoAlaX-M WT H103A, PhoAlaX-M T172Y, EcN1 + N2 WT and EcN1 + N2 Y172V were purified using anion exchange chromatography. Cells were lysed in buffer composed of 50 mM Tris pH 8, 10 mM NaCl and 5 mM β-marcaptoethanol (BME) and spinned at 18,000 × g. For the archaeal proteins the supernatant were heated at 70 °C for 30 min to precipitate the E. coli proteins and then spinned at 18,000 × g. The supernatant was then loaded onto q-sepharose column (Cytiva, USA), which was equillibriated with lysis buffer. Proteins were eluted in buffer composed of 50 mM Tris pH 8, 250 mM NaCl and 5 mM BME. After purification, the fractions containing the protein of interest were pooled, concentrated and subjected to further purification by SEC (Superdex-75) in buffer composed of 50 mM Tris pH 8, 150 mM NaCl and 5 mM BME.

EcAlaRS H568A N-His, EcAlaRS L656Y N-His and EcThrRS WT N-His were overexpressed in E. coli BL21 (DE3). The cells were lysed in buffer composed of 50 mM Tris pH 8.0, 150 mM NaCl, 30 mM imidazole and 5 mM BME. The lysate was loaded onto Ni-NTA column equillibrated with the same buffer. Post loading, the column was washed with lysis buffer and then followed by wash buffer composed of 50 mM Tris pH 8.0, 150 mM NaCl, 30 mM imidazole and 5 mM BME. Protein was eluted with 50 mM Tris pH 8.0, 150 mM NaCl, 250 mM imidazole and 5 mM BME. The fractions containing protein were pooled, concentrated and then subjected to further purification by SEC (Superdex-200) in buffer composed of 50 mM Tris pH 8, 150 mM NaCl and 5 mM BME.

Removal of Zn²⁺ from AlaX-proteins and verification using PAR assay

To remove Zn²⁺ from EcAlaRS WT and PhoAlaX-M WT enzymes, the respective purified protein was treated with 5 mM 1,10-Phenanthroline (final concentration) in buffer composed of 50 mM Tris pH 8, 150 mM NaCl and 1 mM TCEP at 4 °C for over night (~12 h) followed by thorough buffer-exchange with metal-free buffers composed of Tris pH 8, 150 mM NaCl and 1 mM TCEP using 10 kDa MWCO centricon. The metal-free buffers were prepared by passing the buffers through Chelex-100 (BioRad) column.

Level of Zn²⁺ bound to protein was assayed spectroscopically using a chromophoric chelator 4-(2-pyridylazo)-resorcinol (PAR)⁴⁰. Around 50 µM of proteins were treated with 6 M guanidium hydrochloride (GdnHCl) and 50 µM PAR in buffer containing 50 mM HEPES pH 7.5 and subsequently after 1 h incubation at 37 °C, absorbance spectrum in wavelength range 350 nm to 650 nm were recorded.

PAR-based competition assay

The competition assay was performed as mentioned in Kocyla et al., (ref. ⁴²). Various concentration of PhoAlaX-M WT apo, PhoAlaX-M H103A, PhoAlaX-M T172Y and EcN1 + N2 WT were titrated against 100 µM PAR partially saturated with 10 µM of ZnSO₄ in buffer composed of 100 mM HEPES pH 7.4 and 100 mM NaCl on ice. The samples were incubated for ~12 h on ice for equilibration. Absorbance at λ = 492 nm was recorded and the concentration of Zn•(PAR)₂ in each sample was estimated from standard curve of ZnSO₄ and PAR in identical buffer condition. The exchange constant (K_ex) for all the samples were calculated according to Eq. (1):

$${K}_{{ex}}=\frac{{\left[{PAR}\right]}^{2}\times [{Zn} \cdot {Protein}]}{\left[{{Zn} \cdot \left({PAR}\right)}_{2}\right]\times [{Protein}]}$$

(1)

The dissociation constant of the protein samples (Kd^Protein) were calculated using the dissociation constant of Zn•(PAR)₂ (Kd^PAR = 7.08 × 10⁻¹³ M²) based on Eq. (2):

$${{Kd}}^{{Protein}}={{Kd}}^{{PAR}}\times \frac{1}{{K}_{{ex}}}$$

(2)

Thermal shift assay

Thermal shift assay was performed with 10 µg of proteins in buffer composed of 50 mM HEPES (pH 7.5), 10 mM MgCl₂ and100 mM KCl was mixed with 5X SYPRO Orange Protein Gel Stain (Thermo Scientific) and then melting curve was measured in Bio-RAD CFX Opus realtime PCR system.

Crystallisation, X-ray diffraction data collection and structure determination

Purified untagged PfuAlaX-M protein was screened for initial crystallization at 4 °C with Crystal screen 1 and 2 (Hampton Research, Aliso Viejo, CA). Hits from the screen were further optimised using hanging-drop vapour diffusion method in 24-well Iwaki plates. Diffraction-quality crystals were obtained in 0.2 M Sodium Acetate Trihydrate, 0.1 M Sodium Cacodylate Trihydrate pH 6.5, 30% PEG8000. X-ray diffraction data for the PfuAlaX-M crystal was collected at the in-house X-ray facility using FR-E+ SuperBright X-ray generator from Rigaku equipped with VariMax HF optics and R-AXIS IV++ image plate detector. Data processing was performed using iMOSFLM⁸³ and AIMLESS⁸⁴ programmes from the CCP4 suite and structure solution by molecular replacement was done using MOLREP⁸⁵ from the CCP4 suite using PhoAlaX-M structure (PDB: 2E1B) as the search model. The structure was refined using phenix.refine from PHENIX package⁸⁶. Model building was performed using COOT⁸¹. PyMOL Molecular Graphics System, Version 4.6.0 Schrödinger, LLC and UCSF ChimeraX⁸⁷ was used for visualisation.

Biochemical assay

E. coli tRNA^Ala and E. coli tRNA^Thr were prepared by in vitro transcription using MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, USA). These tRNAs were then radio-labelled at 3’-end by incorporating [α-³²P]-ATP at 76^th position using CCA adding enzyme (Ledoux and Uhlenbeck, 2008).

The 3’-end radio-labelled tRNA^Ala was charged with amino acid in a reaction mix containing 50 mM HEPES pH 7.5, 100 mM alanine/300 mM glycine or serine, 100 mM KCl, 10 mM MgCl₂, 2 mM ATP, 1 μM tRNA^Ala and 5 μM E. coli alanyl-tRNA synthetase C666A enzyme incubated at 37 °C for 15 min. The level of aminoacylation on tRNA^Ala for Ala, Gly and Ser were ~50 %. The 3’-end radio-labelled tRNA^Thr was charged with amino acid in a reaction mix containing 100 mM Tris pH 7.5, 2 mM threonine/serine, 200 mM KCl, 100 mM MgCl₂, 2 mM ATP, 1 μM tRNA^Thr, 10 U ml^-1 PPase and 5 μM EcThrRS ΔN1 + N2 enzyme incubated at 37 °C for 15 min. The level of aminoacylation on tRNA^Thr for Thr and Ser were ~50 %.

Deacylation of Ala/Gly/Ser-tRNA^Ala by AlaRS and AlaX-M proteins was performed as mentioned in Pawar et al.⁴⁸. Briefly, deacylation assay of different AlaRS-Ed enzymes reconstituted in buffer composed of 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 100 mM KCl were performed by incubating with 0.2 μM of a given substrate (α-32P–labelled aa-tRNAs) in buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 100 mM KCl, 2.5 mM β-marcaptoethanol (BME) at 37 °C. For deacylation assays to check the effect of Zn²⁺ on enzyme activity, the respective enzymes were incubated with Zinc Acetate for 30 min on ice in buffer composed of 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 100 mM KCl prior to subjecting them to deacylation reaction containing Zinc Acetate. For deacylation assay to check the effect of H₂O₂, the respective enzymes were pretreated with H₂O₂ for 45 min at 37 °C prior to using them for deacylation reaction in buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 100 mM KCl. Deacylation assays involving Zn²⁺ removed apo-enzymes were performed in Chelex-100 (Biorad) treated buffer and water.

Deacylation of Ser-tRNA^Thr by EcThrRS was performed by incubating enzyme with 0.2 μM of substrate in a buffer containing 50 mM Tris (pH 7.5), 10 mM MgCl₂, 100 mM KCl, 2.5 mM BME at 37 °C. For deacylation assays to check the effect of Zn²⁺ on enzyme activity, the respective enzymes were incubated with Zinc Acetate for 30 min on ice prior to subjecting them to deacylation reaction, also containing Zinc Acetate.

Detection of sulfhydryl in proteins by Ellman’s reagent assay

Primary cultures of the BL21 strains expressing PhoAlaX-M WT, PhoAlaX-M H103A, PhoAlaX-M C182A and PhoAlaX-M T172Y proteins were grown overnight at 37 °C and then 1% of it was used to inoculate large scale secondary cultures. Large scale cultures were grown at 37 °C and 200 rpm shaking till OD₆₀₀ reached 0.6 and then the cultures were induced with 1 mM IPTG and then grown for another 3 h at 37 °C and 200 rpm shaking. Cells were harvested and lysed in buffer containing 50 mM Tris pH 8, 20 mM NaCl, 5 mM BME, 5 mM MgCl₂, 0.2 mM Zn(CH₃CO₂)₂, 2.5 mM TCEP and 1 mM PMSF by sonication in ice. Cell lysate was treated with 1% streptomycin sulphate and then spinned at 18,000 × g. The supernatant was heated at 70 °C for 30 min to precipitate the E. coli proteins and then spinned at 18,000 × g. The supernatant was then loaded onto q-sepharose column (Cytiva, USA), which was equillibriated with lysis buffer composed of 50 mM Tris pH 8, 20 mM NaCl, 5 mM BME, 5 mM MgCl₂, 0.2 mM Zn(CH₃CO₂)₂. Proteins were eluted in buffer composed of 50 mM Tris pH 8, 250 mM NaCl, 5 mM BME, 5 mM MgCl₂, 0.2 mM Zn(CH₃CO₂)₂ in gradient mode. After purification, the fractions containing the protein of interest was checked by SDS-PAGE and were pooled, concentrated and subjected to further purification by SEC (Superdex-75) in buffer composed of 50 mM Tris pH 8, 150 mM NaCl, 5 mM BME, 5 mM MgCl₂, 0.2 mM Zn(CH₃CO₂)₂. 10 µl aliquotes of purified proteins were flash frozen in liquified nitrogen and kept in −80 °C till further use. To detect the protein sulfhydryl group, proteins were first buffer exchanged with 50 mM HEPES pH 7.5, 10 mM MgCl₂ and 100 mM KCl to remove the reducing agents. To check the effect of H₂O₂ on the level of protein sulfhydryl, the proteins were treated with 1 mM H₂O₂ for 45 min at 37 °C in buffer composed of 50 mM HEPES pH 7.5, 10 mM MgCl₂ and 100 mM KCl and then buffer exchanged in 10 kDa MWCO centrifugal filter (Amicon) to remove the H₂O₂. Respective proteins were then incubated with 10 mM DTNB (Ellman’s Reagent) in presence of 6 M guanidine hydrochloride in buffer composed of 100 mM NaH₂PO₄ (pH 8.2) and 0.2 mM EDTA for 60 min and then the Absorbance at 412 nm was measured.

Construction of E. coli ∆dtd ∆alaS deletion mutant

E. coli MG1655 ∆dtd strain generated earlier in the laboratory⁴⁸ was transformed with pBAD33 plasmid (CamR, pACYC origin) containing either ^EcalaS^WT or ^EcalaS^H568A between SacI and HindIII sites. These strains were used for making ∆alaS::Kan deletion using P1 phage-mediated transduction⁸⁸. P1 phage used for alaS knockout was generated earlier in the lab⁴⁸.

Spot dilution assays with E. coli MG1655 ∆dtd ∆alaS mutant

Spot dilution assays were performed to check the effect of abrogation of Zn²⁺ binding in AlaRS editing domain on cell viability. For this purpose, E. coli MG1655 ∆dtd ∆alaS mutant complemented with either EcAlaRS WT or EcAlaRS H568A were grown in a minimal medium (Miller, 1992). Cultures were grown until OD ₆₀₀ reached ~0.6 and were 10-fold serially diluted (10⁻², 10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁶). Of each serially diluted sample, 3 µl was spotted on minimal agar plates containing 0.002% L-arabinose, 0.2% maltose as carbon source. 10 mM glycine, 1 mM L-alanine, 3 mM L-serine and 50 µM H₂O₂ were added to the media depending on the experiment. The plates were incubated at 37 °C for 12 to 16 h.

GFP-based mistranslation assay

To monitor GFP fluorescence, MG1655 ΔalaS Δdtd /P_ara::^EcalaS^WT and MG1655 ΔalaS Δdtd/P_ara::Ec alaS^H568A strains expressing gfp^G67A, primary cultures were grown at 37 °C in LB medium containing 0.002% L-arabinose, 100 mg ml⁻¹ ampicillin and 20 μg ml⁻¹ chloramphenicol. 3% inoculum of the primary culture was used to initiate 5 mL secondary culture in 1X minimal salts with 0.2% maltose as carbon source and 0.002% L-arabinose; the secondary culture of both the strains were supplemented with 10 mM glycine and 50 µM H₂O₂ except for the controls. Cells were immobilised on a thin agarose (1.5%) slide and visualised under a Zeiss Axioimager microscope in DIC (Nomarski optics) and EGFP (Fluorescence) mode.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Atomic coordinates for PfuAlaX-M is deposited in the Protein Data Bank under accession number 9IUW. All data needed to evaluate the conclusions in the paper are present in the paper, Supplementary Materials and source data files. Source data are provided with this paper.

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Acknowledgements

J.G. thanks University Grants Commission, India for research fellowship. R.S.N. thanks healthcare theme projects- Fundamental and Innovative CSIR in Science of Tomorrow (FIRST; MLP-0162) and Niche Creation Project (NCP; MLP-0138) of CSIR, India; J.C. Bose Fellowship of SERB, India; and Centre of Excellence Project of Department of Biotechnology, India. We thank Ms. Rukmini Raju and Mr. Mallesham Karsala, X-ray facility CSIR-CCMB for assistance during X-ray diffraction data collection. We thank Dr. Satya Brata Routh for his valuable suggestions related to the work. We thank Dr. Manjula Reddy, CCMB and her lab members for help in generating bacterial knock-out.

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CSIR-Centre for Cellular and Molecular Biology, Hyderabad, 500007, Telangana, India
Jotin Gogoi, Komal Ishwar Pawar, Koushick Sivakumar, Akshay Bhatnagar, Katta Suma, Kezia J. Ann, Sambhavi Pottabathini, Shobha P. Kruparani & Rajan Sankaranarayanan
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
Koushick Sivakumar & Rajan Sankaranarayanan

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Jotin Gogoi
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Komal Ishwar Pawar
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Kezia J. Ann
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Sambhavi Pottabathini
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J.G. and R.S.N. designed the study. J.G., K.I.P., K.S.I., A.B., K.S.U., K.J.A., S.P., S.P.K. performed the experiments. R.S.N. conceived and supervised the study. All the authors analysed the data. J.G. and R.S.N. wrote the manuscript and all the authors reviewed it.

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Correspondence to Rajan Sankaranarayanan.

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Gogoi, J., Pawar, K.I., Sivakumar, K. et al. A metal ion mediated functional dichotomy encodes plasticity during translation quality control. Nat Commun 16, 3625 (2025). https://doi.org/10.1038/s41467-025-58787-2

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Received: 23 August 2024
Accepted: 30 March 2025
Published: 16 April 2025
DOI: https://doi.org/10.1038/s41467-025-58787-2