Fig. 2: Opposing effect of Zn2+ and ROS on activity of homologous AlaRS-Ed and ThrRS-Ed.
From: A metal ion mediated functional dichotomy encodes plasticity during translation quality control
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 ThrRS-Ed involved in activation of water molecule (W1) during catalysis is displaced in presence of Zn2+ and H604 in AfuAlaRS-Ed also adopts a similar position. h Deacylation of Ser-(Ec)tRNAAla by buffer, 50 nM EcAlaRS holo, EcAlaRS holo + 5 mM H2O2; (N = 3). i Graph showing deacylation of Ser-(Ec)tRNAAla by buffer, 50 nM EcAlaRS apo, EcAlaRS apo + 5 mM H2O2. j Schematic diagram showing method to detect oxidised/un-oxidised sulfhydryl in proteins by Ellman’s assay. k Bar plot showing absorbance at λ = 412 nm of Ellman’s reagent treated protein samples indicating level of sulfhydryl group in PhoAlaX-M WT holo, PhoAlaX-M WT apo, PhoAlaX-M C182A and EcThrRS-Ed (N1 + N2); (N = 3). In all the graphs in the panel data are represented as mean ± standard deviation. Statistical analysis in Fig. 2h was done using unpaired two-sided Student’s t test. Source data are provided in the Source Data file.
