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. 2024 Dec 30;15(1):10817.
doi: 10.1038/s41467-024-55183-0.

The mechanism of discriminative aminoacylation by isoleucyl-tRNA synthetase based on wobble nucleotide recognition

Bingyi Chen  1   2 , Fang Yi  1   2 , Zhiteng Luo  1   2 , Feihu Lu  1   2 , Hongwei Liu  3 , Siting Luo  1   2 , Qiong Gu  1 , Huihao Zhou  4   5
Affiliations

The mechanism of discriminative aminoacylation by isoleucyl-tRNA synthetase based on wobble nucleotide recognition

Bingyi Chen et al. Nat Commun. .

Abstract

The faithful charging of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases (AARSs) determines the fidelity of protein translation. Isoleucyl-tRNA synthetase (IleRS) distinguishes tRNAIle from tRNAMet solely based on the nucleotide at wobble position (N34), and a single substitution at N34 could exchange the aminoacylation specificity between two tRNAs. Here, we report the structural and biochemical mechanism of N34 recognition-based tRNA discrimination by Saccharomyces cerevisiae IleRS (ScIleRS). ScIleRS utilizes a eukaryotic/archaeal-specific arginine as the H-bond donor to recognize the common carbonyl group (H-bond acceptor) of various N34s of tRNAIle, which induces mutual structural adaptations between ScIleRS and tRNAIle to achieve a preferable editing state. C34 of unmodified tRNAIle(CAU) (behaves like tRNAMet) lacks a relevant H-bond acceptor, which disrupts key H-bonding interactions and structural adaptations and suspends the ScIleRS·tRNAIle(CAU) complex in an initial non-reactive state. This wobble nucleotide recognition-based structural adaptation provides mechanistic insights into selective tRNA aminoacylation by AARSs.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The ScIleRS·tRNAIle(GAU)·l-Ile complex structure in the editing state.
a Domain diagram of IleRSs from Saccharomyces cerevisiae, Homo sapiens, Pyrococcus horikoshii and Staphylococcus aureus. b Cloverleaf models of tRNAIle(GAU) and tRNAIle(CAU) from Escherichia coli. c Cartoon representation of the overall structure of ScIleRS in complex with tRNAIle(GAU) and l-Ile. ScIleRS is colored the same as the domain diagram, the substrate l-Ile is represented as spheres, and tRNAIle(GAU) is colored in orange with the nucleotides directly interacting with ScIleRS shown in filled rings. d Structural superposition of the ScIleRS·tRNAIle(GAU)·l-Ile complex and the Thermus thermophilus IleRS (TtIleRS) ED·Val-2AA complex (PDB ID: 1WNZ, green) confirms the protein‒tRNA interactions in the editing state revealed by the small-molecule probe Val-2AA. The H-bonds between residues of ScIleRS and nucleotide A76 of tRNAIle(GAU) are shown as black dashed lines. e tRNALeu was modeled to IleRS according to the EcLeuRS·tRNALeu·Leu-AMP complex structure in the aminoacylation state (PDB ID: 4AQ7). The CP core of ScIleRS in the editing state largely overlaps with the acceptor arm of tRNALeu. f In contrast, structural modeling revealed that there is no significant conflict between the acceptor arm of tRNALeu and the CP core of SaIleRS in the SaIleRS·tRNAIle(GAU)·mupirocin complex (PDB ID: 1FFY).
Fig. 2
Fig. 2. Productive tRNAIle binding requires conformational movements at ScIleRS C-terminal domains.
a The C-ter B domain of ScIleRS is stabilized and binds to the elbow of tRNAIle(GAU). b Interactions with the C-ter B domain induce a conformation of the U20 nucleotide of ScIleRS-bound tRNAIle(GAU) opposite to that of SaIleRS-bound tRNAIle(GAU) (PDB ID: 1FFY). c ScIleRS proteins with mutations located far from the active site exhibited similar or comparable activity to that of the wild-type protein in the tRNA-independent pre-transfer editing assay. Data are presented as means ± SD (n = 3 independent experiments). d Most ScIleRS variants partially or completely lost the tRNAIle isoleucylation activity as measured by tRNA-dependent ATP consumption assay. EctRNAIle(GAU) overexpressed in E. coli cells was utilized in this assay. Data are presented as means ± SD (n = 3 independent experiments). e The EMSA assay result revealed that tRNAIle binding ability of ScIleRSΔCB is weaker than that of wild-type ScIleRS. The in vitro transcript of tRNAIle(GAU) was used in this assay. Similar results were observed in two independent experiments. f The aminoacylation activity of ScIleRS against in vitro transcribed tRNAIle(GAU) and its variants with G19C, G18C&C56A or U55A mutations. All the variants presented significantly lower isoleucylation than that of the wild-type tRNAIle(GAU). Data are presented as means ± SD (n = 3 independent experiments). g, h Structural comparison of the tRNAIle(GAU)-bound ScIleRS with the tRNA-free ScIleRS (PDB ID: 7D5C, colored in gray) (g) and apo ScIleRS (AlphaFold DB: AF-P09436-F1, colored in pink) (h) indicated the conformational changes in the C-terminal domains and ABD of ScIleRS upon tRNAIle binding.
Fig. 3
Fig. 3. Recognition of A35/U36 by the ABD of ScIleRS.
a Electrostatic surface potential of the positively charged cavity formed by the ABD, C-ter A, and C-ter C domains for binding the anticodon loop of tRNAIle(GAU). b Base-specific interactions between the anticodon loop of tRNAIle(GAU) and the ABD of ScIleRS. c As shown in the SaIleRS·tRNAIle(GAU)·mupirocin complex structure (PDB ID: 1FFY), the ABD of SaIleRS only forms base-specific interactions with A35 of tRNAIle(GAU). d Different conformations of the anticodon loop between SaIleRS-bound and ScIleRS-bound tRNAIle(GAU) molecules. The nucleotides U33, G34 and U36 in the SaIleRS-bound tRNAIle(GAU) clash with the C-ter C domain of ScIleRS.
Fig. 4
Fig. 4. Structural mechanism of N34 recognition by the C-ter C domain of eukaryotic/archaeal-type IleRS.
a The binding of G34 to the C-ter C domain of ScIleRS. The top panel shows a zoomed-in view of the G34 binding site of the ScIleRS·tRNAIle(GAU)·l-Ile complex. Direct and water-mediated H-bonding interactions are shown as black dashed lines. The bottom panel is the 2D presentation of G34–residues interactions, and the direct H-bonds are shown as orange dashed lines. The water-mediated H-bonding interactions have been omitted in the 2D presentation. b 2D presentations of the modeling of A34 and I34 at the N34 binding site of ScIleRS. A34 is unable to interact with the N34 binding residues due to the lack of appropriate H-bond acceptors or donors, so it is poorly recognized by ScIleRS. In contrast, after deamination, I34 can bind to these residues like G34, and be well recognized by ScIleRS. c Modeling of U34 and Ψ34 at the N34 binding site of ScIleRS. d Modeling of C34 and agm2C34 at the N34 binding sites of ScIleRS and PhIleRS, respectively.
Fig. 5
Fig. 5. The ScIleRS·tRNAIle(CAU)·l-Ile complex is stalled in a non-reactive state due to the lack of key N34 interactions.
a The aminoacylation activities of ScIleRS and SaMetRS against in vitro transcribed tRNAIle(GAU), tRNAIle(CAU) and tRNAIle(CAU) C34G variant. Data are presented as means ± SD (n = 3 independent experiments). b EMSA revealed that in vitro transcribed tRNAIle(CAU) can still form a complex with ScIleRS, although it is slightly weaker than in vitro transcribed tRNAIle(GAU). Similar results were observed in two independent experiments. c The overall structure of the ScIleRS·tRNAIle(CAU)·l-Ile complex. The anticodon loop of tRNAIle(CAU) is dynamic because of the lack of interactions with ScIleRS, and the acceptor stem of tRNAIle(CAU) binds to the back of the ED. d Binding of the acceptor stem of tRNAIle(CAU) to the back of ScIleRS ED. The 3’ CCA end was invisible in the electronic density map. e Structural comparison between the reactive and non-reactive ScIleRS·tRNA complexes revealed an approximately 25° rotation between tRNAIle(GAU) and tRNAIle(CAU). f The C-terminal domains of tRNAIle(CAU)-bound ScIleRS adopt a conformation generally similar to that of ScIleRS in the tRNA-free state except for the distal C-ter C domain, but not to that of tRNAIle(GAU)-bound ScIleRS.
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