Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3

Abstract

The essential deamination of adenosine A₃₄ to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A₃₄ deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.

Fig. 1. The structure of TbADAT2/3 heterodimer bound to tRNA^Thr_CGU.

a Schematic representation of the protein subunits: ADAT2 CDA in salmon, ADAT3 N-terminal domain in blue, ADAT3^CDA in teal. Portions with traced atomic model as solid colors and non-resolved linkers as dotted lines. Important sequence motifs are annotated at their relative positions. Zn²⁺ coordinating residues are indicated by yellow triangles, the active-site glutamate / pseudo-active-site valine by a red and gray star, ‘gate’ residues of ADAT2 with a blue triangle. Conserved positive charges of the KR-motif annotated as blue “+“ signs. b Structural alignment of Tb, yeast and mouse ADAT2 and ADAT3 CDA domains and Staphylococcus aureus TadA. The sequences for human (Hs) ADAT2/3 included (MmADAT2/3 PDB: 7NZ7, scTAD2/3 PDB:7BV5, saTadA PDB: 2B3J). Structural elements of Tb are annotated (ADAT2 above, ADAT3 below) with α-helices as cylinders and β-sheets as arrows. Zn²⁺ coordinating residues are indicated by yellow triangles, the active-site glutamate / pseudo-active-site valine by a red and gray star, ‘gate’ residues of ADAT2 with a blue triangle and the active-site histidine of ADAT3 with a teal triangle. ADAT2^C is boxed in blue with annotation of the conserved positive charges of the KR-motif (“+“ signs). Grayed out letters indicate non-resolved loops in our structure and ADAT2^C residues not resolved in previous crystal structures. An ADAT3 conserved loop is annotated as a teal box. Other colors and boxes within the alignment represent relative conservation above 70%. c Four views of the final non-b-factor-sharpened cryoSPARC map that was used for modeling (presented at a level of 0.09) with atomic model. Colors as above. d Cartoon model of ADAT2/3 bound to tRNA. Colors as in 1 A. Zn²⁺ atoms in yellow, tRNA in black with only anticodon loop bases represented as slabs, the reactive C₃₄ nucleotide is annotated as C*. Gray arrows point to the ADAT3 N-terminal domain double linker.

Fig. 2. The interaction of the tRNA anticodon-stem loop with ADAT2/3.

a The anticodon loop (ACL) bound to TbADAT2/3 (black) is distorted with respect to free tRNA (brown; PDB: 1EHZ) but resembles the ACL bound to bacterial TadA (gray; PDB: 2B3J), all cartoon-stick representation. b A deep cleft in the dimer interface accommodates the ACL. Colors as in Fig. 1, ADAT2/3 model as surface representation, tRNA as cartoon with anticodon loop bases shown. c The deformed ACL bound to the TbADAT2/3, backbone in cartoon and bases in stick representation. d Schematic overview of the anticodon stem loop (ACSL) and its interactions with TbADAT2/3. Zinc coordinating site highlighted in yellow. e–g Comparison of the active-site pockets of Tb and MmADAT2/3 and TadA in the same orientation: e TbADAT2/3 with tRNA cytosine-base shown; f apo-MmADAT2/3 (PDB: 7NZ7), with the conserved H298^MmADAT3 as the only ADAT3 residue present in the active site; g bacterial SaTadA (PDB: 2B3J) with nebularine bound, with M92^SaTadA훃 residue contributed by the second protomer of the homodimer. Residue side chains colored by heteroatom with zinc atoms in yellow.

Fig. 3. The tRNA anticodon loop passes through a molecular RY-gate in ADAT2.

a Superposition of the tRNA-bound gate in TbADAT2/3 and the apo-gate in MmADAT2/3 (PDB: 7NZ7). The side chain of R159^TbADAT2 is represented as flexible due to its poor side chain density. b The corresponding positions in bacteria deaminase TadA, RNA bound versus unbound (PDB: 2B3J and 1WWR, respectively). c Fold change of the observed rate constants of mutants to wild-type (k_rel = k_obs mutant/k_obs WT). Graph values calculated from three independent replicates. All mutants are of TbADAT2 co-expressed with TbADAT3. n.d. denotes no detectable activity after 24 h assay incubation. k_obs values obtained from single-turnover kinetic assays (n≥3). Source data are provided as a Source Data file.

Fig. 4. The ADAT2 C-terminal region including the KR-motif.

a The C-terminus of ADAT2 embraces nucleotides 35 and 37 and KR-loop residues (K or R) provide additional contacts with the anticodon-stem loop via phosphate backbone interactions at residues 28–31. A cartoon representation of the ADAT2 C-terminus and tRNA is shown with the final non-b-factor-sharpened cryoSPARC map. Due to the limited resolution, we refrain from showing side chains in the ADAT2^C model. b The same region of ScTAD2/3 (PDB: 7BV5), MmADAT2/3 (PDB: 7NZ7), and RNA bound SaTadA (PDB: 2B3J). The flexible unmodeled loop of ScADAT2 is shown as a dotted line.

Fig. 5. ADAT3 N-terminal domain probes the three-dimensional structure of the tRNA.

a Two views of the ADAT3^N fitted in the post-processed EM map, in blue, with ‘A’ and ‘B’ regions as the two contact points of our model with tRNA, as indicated in C. b ADAT3^N including linkers (residues 4–172); surface model colored by vacuum electrostatic potential, positive charges in red and negative charges in blue, with D-Arm of tRNA in a black cartoon representation. Residues identified to play a role in tRNA binding are annotated in black, in olive from and in yellow from. (c) Schematic of the tRNA with interaction contacts. Anticodon-stem loop interactions are represented as a summary of the ones indicated in Fig. 2D. The two ADAT3^N contact points, ‘A’ and ‘B’ are indicated in blue as above.

Fig. 6. The hypothetical multi-step tRNA recognition mechanism of ADAT2/3.

a Ligand-free enzyme state: in the free ADAT2/3 heterodimer ADAT2^C is disordered and ADAT3^N is flexibly linked. b Initial capture: the RNA recognition motifs in ADAT3^N and in ADAT2^C capture RNA; tRNAs are progressively guided by the extension of favorable interactions towards the anticodon binding cleft; hereby non-tRNA architectures will be rejected. c Anticodon loop insertion into the active site: The anticodon loop enters the active-site cleft through the RY-gate, and ADAT2^C folds into the major groove of the anticodon loop to position the ligand for the reaction. d Substrate release: Detachment of the individual RNA-binding motifs initiates tRNA release.