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

Figures

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  Fig. 1. Zn2+ in AlaRS-Ed is dispensable for structure and proofreading activity. a Graph showing deacylation of Ala-(Ec)tRNAAla by buffer (black square), EcAlaRS holo (black triangle) and EcAlaRS apo (black circle); (N = 3). b Graph showing deacylation of Ser-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS holo (black triangle) and 50 nM EcAlaRS apo (black circle); (N = 3). c Graph showing deacylation of Gly-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS holo (black triangle) and 50 nM EcAlaRS apo (black circle); (N = 3). d Structural superposition of PfuAlaX-M holo (PDB: 9IUW), PhoAlaX-S holo (PDB: 1WXO) (Sokabe et al.27), PhoAlaX-S apo (PDB: 1V7O) (Sokabe et al.27) and AfuAlaRS cis-Ed holo (PDB: 2ZTG) (Naganuma et al.51) showing good overlap of structures with average r.m.s.d. of ~1.3 Å over 131 Cα atoms (Supplementary Fig. 3i). e Zoomed in view of superposed AlaRS-Ed variant structures showing HXXXH, CXGXH motifs coordinating a Zn2+ ion. f Zoomed in view of the overlap of AfuAlaRS cis-Ed holo (PDB: 2ZTG) and PhoAlaX-S apo (PDB: 1V7O) structures with r.m.s.d. of ~1.4 Å over 137 Cα atoms and shows no notable distinction in positioning of Zn2+ ligand residues in HXXXH, CXGXH…

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  Fig. 2. Opposing effect of Zn2+ and ROS on activity of homologous AlaRS-Ed and ThrRS-Ed. a Sequence logo showing conservation of HXXXH and CXGXH Zn2+ binding motifs in AlaRS cis-Ed, AlaXps and ThrRS-Ed (N2) based on structure-based sequence alignment (Supplementary Fig. 2). Sequence numbering is based on EcAlaRS, PhoAlaX-M and EcThrRS. b Structural superposition of EcThrRS-Ed (N2) (PDB: 1TKE) (Dock-Bregeon et al.30) and AfuAlaRS cis-Ed (PDB: 2ZTG). Inset: zoomed in view of the catalytic site showing presence or absence of Zn2+ in AlaRS-Ed and ThrRS-Ed. c Titration of PAR•Zn2+ with PhoAlaX-M WT apo and EcN1 + N2 WT (ThrRS-Ed); (N = 3). d Deacylation of Ser-(Ec)tRNAThr by buffer, 50 nM EcThrRS, EcThrRS + 10 µM Zn2+, EcThrRS + 50 µM Zn2+ and 50 nM EcThrRS + 100 µM Zn2+; (N = 3). e Deacylation of Ser-(Ec)tRNAAla by buffer, 50 mM Zn2+, 50 nM EcAlaRS, EcAlaRS + 10 mM Zn2+, EcAlaRS + 50 mM Zn2+; (N = 3). f Structure of EcThrRS-Ed (PDB: 1TKY) showing C182 involved in substrate binding. g Structural superimposition of substrate analogue Ser-3AA bound EcThrRS-Ed (PDB: 1TKY) in blue, Zn2+ bound SaThrRS-Ed structure (PDB: 1NYR) in green and AfuAlaRS cis-Ed (PDB: 2ZTG) in grey showing that H73 in…

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  Fig. 3. Zn2+ protects AlaRS editing activity from ROS in vivo. a Schematic diagram showing HXXXH and CXGXH Zn2+ binding motifs, residues marked with symbol ‘#’ indicates residues whose mutation affects activity and symbol ‘*’ represent residues with minor consequence on activity upon mutation. b Deacylation of Ser-(Ec)tRNAAla by buffer (black diamond), 50 nM EcAlaRSH568A (purple diamond), 50 nM EcAlaRSH568A + 50 µM H2O2 (green diamond), 50 nM EcAlaRSH568A + 100 µM H2O2 (blue diamond), 50 nM EcAlaRSH568A + 1 mM H2O2 (pink diamond), 50 nM EcAlaRSH568A + 5 mM H2O2 (orange diamond); (N = 3). c Graph showing deacylation of Ala-(Ec)tRNAAla by buffer (black diamond), 50 nM EcAlaRSH568A (black triangle); (N = 3). d Bar plot showing level of sulfhydryl group in PhoAlaX-M H103A using Ellman’s reagent; (N = 3). e Spot dilution-based growth assay of E. coli MG1655 Δdtd ΔalaS strains complemented with either alaSWT or alaSH568A in M9 minimal agar plate under conditions: control, 3 mM Ser, 3 mM Ser + 50 µM H2O2, 3 mM Ser + 50 µM H2O2 + 10 mM Ala, 10 mM Gly, 10 mM Gly + 50 µM H2O2, 10 mM Gly + 50 µM H2O2 + 10 mM Ala. Representative plates of three independent experiments are shown. f A GFP-based…

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  Fig. 4. Structures reveal determinant of difference in Zn2+ binding by AlaRS-Ed and ThrRS-Ed. a Structural superimposition of AfuAlaRS cis-Ed (PDB: 2ZTG) in grey and EcThrRS-Ed (PDB: 1TKE) in blue showing good overlap of the structures with r.m.s.d. of ~ 2.4 Å over 135 Cα. b Zoomed in view of the catalytic pocket showing HXXXH and CXGXH motifs displaying proper steric organisation of the coordination sphere in AlaRS-Ed for coordinating Zn2+, but not in ThrRS-Ed, as reflected by the difference in inter-ligand distances. The distorted steric arrangement of the ligand residues arises from the distinct positioning of the loop spanning CXGXH in ThrRS-Ed is due to the Y173, which in case of AlaRS-Ed is an aliphatic amino acids V/I/L/T. c Sequence logo showing conservation of Y173 in ThrRS-Ed in organisms across bacteria, archaea and eukarya, whereas in case of AlaRS-Ed the corresponding residue position is occupied by aliphatic side chain amino acids V/I/L/T (Supplementary Fig. 7a). d Backbone trace view of superposition of all the structures of AlaRS-Ed and ThrRS-Ed available in PDB database with resolution ≥2.5 Å, showing distinct positioning of the loop region spanning CXGXH motif in both…

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  Fig. 5. Implanting ThrRS-Ed determinant in AlaRS-Ed affects its Zn2+ binding and ROS resistance of activity. a Schematic diagram showing PAR-based competitive assay for assessing Zn2+ binding strength. b Graph showing absorbance spectra across λ = 450 nm to 550 nm of PAR assay samples: 50 µM EcAlaRS WT with GdnCl (red), 50 µM EcAlaRS WT without GdnCl (green), 50 µM EcAlaRS L656Y with GdnCl (black) and 50 µM EcAlaRS L656Y without GdnCl (purple); (N = 3). c Graph showing absorbance spectra across λ = 450 nm to 550 nm of PAR assay samples: 50 µM Zinc acetate (blue), 50 µM EcN1 + N2 WT with GdnCl (red), 50 µM Ec N1 + N2 WT with GdnCl (green); (N = 3). d Deacylation of Ser-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS WT (black triangle), 50 nM EcAlaRS WT + 5 mM H2O2 (black circle), 50 nM EcAlaRS WT + 10 mM H2O2 (black diamond); (N = 3). e Deacylation of Ser-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS L656Y (black triangle), 50 nM EcAlaRS L656Y + 5 mM H2O2 (black circle), 50 nM EcAlaRS L656Y + 10 mM H2O2 (black diamond); (N = 3). f Bar plot showing level of sulfhydryl group in PhoAlaX-M T172Y upon treatment with H2O2 using Ellman’s reagent; (N = 3). g Spot dilution-based growth…

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  Fig. 6. Ancient divergence between ThrRS-Ed and AlaRS-Ed. a Maximum likelihood phylogenetic tree of AlaRS cis-Ed, AlaX-L, AlaX-M, AlaX-S and ThrRS-Ed protein sequences from representative organisms belonging to three domain of life reconstructed using IQ-TREE web server (Nguyen et al.79). The bootstrap branch support ≥90 are indicated by black circle. b A schematic diagram of AlaRS-Ed showing canonical or the primary (1°) site of Zn2+ binding formed by HXXXH/CXGXH motifs and a hypothesised secondary (2°) transient Zn2+ binding site, shown as blue shaded area, where the Zn2+ migrates to upon substrate binding, depicted by red dashed arrow. The Zn2+ ion is hypothesised to migrate between the 1° and 2° sites in cycle, in sync with the catalysis cycle of AlaRS-Ed comprising of substrate binding, substrate hydrolysis and product release. c A schematic model depicting the pre-LUCA evolutionary divergence of AlaRS-Ed and ThrRS-Ed in terms of Zn2+ binding strength by virtue of a difference in a residue that is part of hydrophobic shell surrounding the coordination sphere, leading to distinct sensitivity of their activities toward ROS. The distinct sensitivity of AlaRS-Ed and ThrRS-Ed activities…