Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

Abstract

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

Fig 1. DTD binds and deacylates achiral glycine.

(a) Excerpts of overlay of 2D ¹⁵N-¹H Transverse Relaxation Optimized Spectroscopy obtained with 0.2 mM PfDTD (black) when titrated with 3 mM each of L-Ala3AA (green), Gly3AA (red), and D-Ala3AA (blue). Arrows indicate chemical shift perturbations. (b) Deacylation of L-Ala-tRNA^Ala and Gly-tRNA^Gly by buffer (blue circle), 50 nM EcDTD (brown triangle), and 500 pM PfDTD (red square). Error bars indicate one standard deviation from the mean. The underlying data of panel (b) can be found in S1 Data.

Fig 2. Glycine misediting by DTD is a universal phenomenon.

(a) Structure-based multiple sequence alignment of DTD from various organisms highlighting the variant residues in the active site. Residues marked with stars are within 6 Å radius of D-Tyr moiety of D-Tyr3AA. Amino acids enclosed in green boxes are varying in DTDs across different organisms. DTDs from organisms indicated by black arrowheads have been tested for biochemical activity in the current study. (b) Non-conserved residues in the chiral proofreading site of various DTDs—Plasmodium falciparum (green; PDB id: 4NBI), Escherichia coli (violet; PDB id: 1JKE), Leishmania major (cyan; model), Drosophila melanogaster (yellow; model), and Danio rerio (pink; model). (c) Deacylation of Gly-tRNA^Gly by buffer (blue circle), 50 nM LmDTD (green circle), 50 nM DmDTD (red square), and 50 nM DrDTD (brown triangle). Error bars indicate one standard deviation from the mean. The underlying data of panel (c) can be found in S1 Data.

Fig 3. Glycine binding mode in the chiral proofreading site of DTD.

(a) Co-crystal structure of PfDTD with Gly3AA showing the capture of the ligand. (b) Comparison of PfDTD+Gly3AA complex with PfDTD+D-Tyr3AA complex (PDB id: 4NBI) showing the flipped orientation of the α-NH₂ group of Gly3AA. (c) Network of interactions of Gly3AA with active site residues of PfDTD. Residues indicated by * are from the dimeric counterpart.

Fig 4. Mechanistic design principle of the active site of DTD.

Schematic showing the relative orientations of different groups attached to Cɑ of the ligand when viewed down Cα–C bond (centre of the wheel). The carboxyl plane of the ligand, represented as a white dashed line, with the two oxygen atoms at 0° and 180°, has been taken as the reference for calculating the angular sweeps. The top two wheels and the middle wheel represent each group attached to the chiral centre individually, while the bottom two wheels and the middle wheel represent each of the three ligands—L-alanine (with methyl and amino groups), glycine (with amino group only), and D-alanine (with methyl and amino groups). The following colour coding for different groups has been used: H: dark green, NH₂: blue, CH₃: brown. The delineation of regions is based on the following distance criteria from Gly149 O or Pro149 O for each group: H, 2.0 Å to 3.0 Å; NH₂, 2.5 Å to 3.5 Å; CH₃, 3.0 Å to 4.0 Å [34]. Regions falling within the range are favoured (green: region of interaction), above the range are allowed (yellow: region of no interaction), and below the range are disallowed (red: region of steric clash). In the case of L-alanine, the amino acid can be squeezed in the pocket with the methyl group positioned at 3.0 Å from the Gly149 O at 180° and the amino group at 2.5 Å from Pro150 O at 60°; hence, the possible allowed region for L-alanine has been shaded grey, and any other positioning of the methyl group would result in more serious clashes. Calculations are based on protein-based superimposition of all the monomers of PfDTD+Gly3AA complex and the average Cɑ, C, and O positions of the glycyl moiety. For distance measurements, all the observed orientations of Gly-cisPro motif are taken into consideration, thereby accounting for the plasticity of the active site.

Fig 5. EF-Tu confers protection on Gly-tRNA^Gly.

(a) Deacylation of Gly-tRNA^Gly in the presence of unactivated EF-Tu (dark blue circle), activated EF-Tu (light blue circle), unactivated EF-Tu and 5 nM EcDTD (red square), activated EF-Tu and 5 nM EcDTD (pink square), unactivated EF-Tu and 10 nM EcDTD (dark green triangle), activated EF-Tu & 10 nM EcDTD (light green triangle), unactivated EF-Tu and 20 nM EcDTD (dark brown diamond), and activated EF-Tu and 20 nM EcDTD (light brown diamond). (b) Deacylation of D-Tyr-tRNA^Tyr in the presence of unactivated EF-Tu (light blue circle), activated EF-Tu (pink square), unactivated EF-Tu and 5 nM EcDTD (dark blue circle), activated EF-Tu and 5 nM EcDTD (red square). Error bars indicate one standard deviation from the mean. The underlying data can be found in S1 Data.

Fig 6. DTD overexpression causes cellular toxicity.

(a) Spot dilution assay of E. coli K12Δdtd::Kan complemented with pTrc99 empty vector, EcDTD/PfDTD wild-type, or EcDTD A102F/PfDTD A112F. (b) Growth curve of E. coli K12Δdtd::Kan complemented with pTrc99 empty vector uninduced (light blue circle) or induced (dark blue circle), PfDTD wild-type uninduced (pink square) or induced (red square), and PfDTD A112F uninduced (light green triangle) or induced (dark green triangle); induction with 0.1 mM IPTG. Error bars indicate one standard deviation from the mean. (c) Spot dilution assay of E. coli K12Δdtd::Kan complemented with pTrc99 empty vector or PfDTD wild-type. The top panel represents uninduced cultures (i.e., 0 mM IPTG), whereas the bottom panel corresponds to cultures induced with 0.1 mM IPTG. The cultures are unsupplemented or supplemented with 25 mM glycine or with 150 mM L-alanine. The underlying data of panel (b) can be found in S1 Data.

Fig 7. DTD is an RNA-based catalyst.

(a) Deacylation of Gly-tRNA^Gly(2'-dA76) and Gly-tRNA^Gly(2'-FdA76) by buffer (blue circle), 5 μM EcDTD (brown triangle), and 5 μM PfDTD (red square). Error bars indicate one standard deviation from the mean. (b) Schematic showing the space (catalytic chamber) in the active site of PfDTD, which can accommodate a water molecule (catalytic water, depicted as red sphere), although the water molecule is not observed in the crystal structure. Residues indicated by * are from the dimeric counterpart. (c) Mass spectrometry analysis of the deacylation product (amino acid) after carrying out deacylation of D-Tyr-tRNA^Tyr with PfDTD in H₂O¹⁸. The underlying data of panel (a) can be found in S1 Data.

Fig 8. Model for protection of Gly-tRNA^Gly by EF-Tu.

In the cell, the aminoacyl-tRNA pool comprises mostly L-aminoacyl-tRNAs, some Gly-tRNA^Gly, and very few D-aminoacyl-tRNAs (central circle). L-aminoacyl-tRNAs are not acted upon by DTD and, hence, no editing (top left). DTD efficiently decouples D-amino acids mischarged on tRNAs (chiral proofreading), even in the presence of abundant EF-Tu (top right). Gly-tRNA^Gly can be edited by DTD in the absence of EF-Tu (misediting; bottom left), which is, however, effectively prevented by EF-Tu (protection from misediting; bottom right). Under conditions in which DTD levels are relatively high, i.e., DTD is overexpressed, protection is relieved, leading to glycine misediting and cellular toxicity (bottom middle).