Fig 1.
Cyclic N6-threonylcarbamoyladenosine (cyclic t6A, ct6A) hypermodification in tRNA.
a Schematic cloverleaf representation of tRNALysUUU showing the location of the ct6A modification at A37. b TcdA catalyzes the ATP-dependent cyclization of t6A into ct6A in tRNAs with ANN anticodons in bacteria, yeasts, fungi and plants, which occurs mainly at the A37 position of the anticodon stem loop (ASL) of the tRNA molecule. (c) Recognition of the AAA codon on mRNA by ASL of tRNALysUUU involves interactions between t6A37 and A1 from the AAA codon (PDB 1XMO).
Table 1.
Crystallographic data processing and refinement statistics.
Fig 2.
X-ray crystallographic structure of the binary complex TcdA at 1.77-Å resolution in complex with ATP.
a Ribbon representation of the TcdA homodimer structure in two orientations related by a 90° rotation depicting each subunit in a different color (green or wheat). Two K+ and one Na+ cations are shown as violet and yellow spheres, respectively. The Na+ ion is located right at the homodimeric interface and its coordination sphere comprises amino acid residues from both monomers and water molecules. ATP is bound in a conserved surface pocket (shown as a space-filling model in CPK colors). b Description of secondary structural elements of TcdA monomer. Helices are shown in gold (α-helices are labeled H1-H8 and 310 helices H310a-d), strands in white (B1-B7), and loops in light cyan. c Annotated schematic representation of TcdA topology. Helices and strands are depicted as in b, except for 310 helices which are indicated with purple lines and labeled a-d. K+ and Na+ ions are shown as blue and yellow circles. Dashed lines delimit regions that interact with K+, and structures involved in TcdA dimerization are marked with dark blue asterisks.
Fig 3.
X-ray electron density maps of the ATP- and AMP-binding pocket and metal coordination spheres.
a Detailed view of the ATP-binding site of TcdA. Residues that interact with ATP are labeled and shown as sticks and atom colors (C atoms are in subunit colors, as in Fig. 1). 2mFo-DFc electron density map is depicted around the ATP substrate at 1.5 σ contour level. b Detailed view of AMP bound in the active site. Representation and electron density map as in (a). c, d Metal coordination spheres of K+ (c) and Na+ (d) cations. Interacting residues are labeled and shown as sticks in atom colors (C atoms are in subunit colors), and coordinating water molecules are shown as red spheres. Metals and their coordination spheres are shown in 2mFo-DFc electron density (grey). The anomalous Fourier map calculated from a long wavelength (1.89 Å) dataset at 2.4-Å resolution is shown for K+ (c) in purple; in contrast, Na+ has no anomalous signal at that wavelength, enabling the accurate discrimination between K+ and Na+ cations in the structure.
Fig 4.
Molecular surface representation of a TcdA dimer in two orientations (a) related by a 90° rotation around a horizontal axis. The location of the ATP binding pocket and the interfacial Na+ ion are labeled. Amino acid residues that contribute to the extended, flat dimer interface are mapped onto the molecular surface in green and labeled. b Close-up of the symmetric helical bundle (H3-H4). Key hydrogen bonding and charge interactions are shown as dashed lines. c Helix H1 participates in the dimer interface through hydrogen bonding and van der Waals interactions with H3 from the same chain and H310a from the opposite monomer.
Fig 5.
Structural comparison of TcdA with the homologous E1-like activating enzymes.
a Ribbon representation of TcdA structural homologs found with PDBeFold [51] with structural similarity Q-scores higher than 0.28 (the highest Q-scores are 0.49 with MoeB and 0.46 with ThiF). All structures were superimposed onto TcdA and are shown in similar orientations for comparison: MoeB (PDB 1JWB), ThiF (PDB 1ZFN), MccB (PDB 3H9J) and UBA5 (PDB 3H8V). E1-like core domain is shown in grey; the long α-helix that is structurally equivalent to TcdA α8 is in orange; metal binding sites are in protein-specific colors; K+ ions are in purple, Na+ ions in yellow, and Zn2+ in grey. b Schematic representation of the domain architecture of the E1-like enzymes superimposed in (a), with equivalent color coding; bs, binding site; the sequence position corresponding with disordered loops is indicated by dashed lines and marked with start and end residues. The C-terminal α8 helix in TcdA is structurally equivalent to the α-helix immediately following the E1-like domain in the other enzymes. c Structure-guided multiple sequence alignment of the E1-like domain with overlaid secondary structure from TcdA. Functionally important residues and motifs are annotated as follows: bold underline, P-loop residues; black asterisks, residues in direct contact with ATP; and blue asterisks, residues from helix H310a that make water-mediated contacts with the nucleotide. Conserved residues are shown in shaded colors.
Fig 6.
TcdA interacts transiently with the sulfur-acceptor SufE-like protein CsdE.
a NMR 1H-15N NOESY spectrum of doubly labeled 13C,15N-CsdE in presence (blue) or absence (black) of unlabeled TcdA. Significant shifts in the position of specific NOESY resonance peaks for CsdE residues are labeled. b The position of the affected amino acid residues is mapped onto the NMR structure of CsdE (PDB 1NI7) in ribbon (left) and surface (right) representations. c Average chemical shift (Δδ) of 13C,15N-CsdE upon binding to TcdA as measured from the NOESY spectrum in a plotted against the CsdE amino acid sequence.
Fig 7.
a SV-AUC experiments demonstrate that TcdA contains a tRNALys binding domain. Sedimentation coefficient (S) continuous distributions, c(S), plotted for TcdA, tRNALys, and the TcdA-tRNALys complex. Representative sedimentation scans and residuals after fitting are shown. For each c(S) distribution, the estimated molecular mass and the smallest mass compatible with the experimental results are indicated. b Absorbance at 280 nm plotted against the AUC column length for SE-AUC experiments for TcdA, tRNALys, and TcdA-tRNALys. Residuals after fitting are also shown. c Electrophoretic mobility shift assay (EMSA) provides evidence for the physical binding of TcdA to tRNALys. Samples were run over a 8% TBE-PAGE gel at 4°C for 1 h to fully resolve tRNALys from TcdA-tRNALys bands. TcdA does not enter the gel when tRNALys is not added, most likely due to its intrinsically basic isoelectric point, pI (theoretical pI = 8.8). tRNALys alone migrates as the fastest migrating band. When 5 M tRNALys is added to 3 μM or 15 μM TcdA, protein-tRNALys complexes develop that migrate behind free tRNALys. An arrow indicates the position of the retardation band containing the most abundant TcdA-tRNALys complex (2:2 stoichiometry), and an asterisk marks the position of a slower migrating TcdA-tRNALys complex (most likely, 2:1 stoichiometry).
Fig 8.
Solution structure of TcdA-tRNALys(UUU).
a SAXS data for TcdA-tRNALys was measured using an online HPLC setup to separate complex from excess free tRNA. The graph shows a plot of the SAXS intensity at zero angle, I(0) (left axis, curve represented as a solid black line), and of the radius of gyration, Rg (on the right axis), versus data-collection frames. Frames 253–263 (green line) were merged and used for shape restoration of the TcdA-tRNALys(UUU), and frames 304–310 were used for the control reconstruction of the tRNALys(UUU) shape. b Best model calculated for the TcdA-tRNALys(UUU) complex overlayed by the ab initio SAXS shape calculated with DAMMIF. The crystal structure of TcdA-ATP is represented in green cartoon and the tRNA is depicted with its main chain as a gold ribbon and the bases as ladders. The fit (red line) to the experimental SAXS data (blue points), calculated with CRYSOL, χ2 and residuals are shown. c Like in (b), for free tRNALys(UUU). In this case, the model is a rigid-body fit of the tRNA structure into the ab initio SAXS envelope.
Fig 9.
a Electrostatic potential molecular surface calculated with APBS (Adaptive Poisson-Boltzmann Solver) [55] and rendered with PyMOL (www.pymol.org) [56]. Two views are shown that are related by a 90° rotation around a horizontal axis. The interfacial Na+ cation is depicted as a yellow sphere. b tRNA is modeled on the basis of the SAXS data for the TcdA-tRNALys(UUU) complex bound to the two outer rims of TcdA (represented as in a), where most of the positively charged surface is found. The two tRNA molecules bind to spatially separated and independent surface patches in a symmetric arrangement. The modified ct6A37 nucleotide is shown in cyan. c Detailed view of the TcdA-tRNA binding mode. TcdA is represented as in (a), with Cys66 sulfur atom shown as a green sphere. The tRNA molecule on the front inserts its anticodon-stem loop into the ATP-binding pocket (in spheres and CPK colors), with ct6A37 (cyan) facing the catalytic site.
Fig 10.
Interaction network for ct6A37-tRNAANN modification.
TcdA (surface representation, monomer chains in green and wheat colors) interacts transiently but specifically with the sulfur acceptor CsdE (in grey, with TcdA-binding surface patches in blue and Cys61 in yellow), linking with the CsdA-CsdE cysteine desulfurase system and sulfur trafficking, which are known to be required for ct6A37 synthesis in vivo [16]. The CsdE-TcdA transient interaction is represented by a grey double-headed arrow. TcdA interacts with tRNAANN (KD in the μM range) in a 2:2 complex that harbors ATP-dependent t6A37 dehydratase activity. The ct6A37 hypermodification is important for decoding efficiency and translation fidelity.