Structure-function analysis of tRNA t6A-catalysis, assembly, and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

Abstract

The universal N⁶-threonylcarbamoyladenosine (t⁶A) at position 37 of tRNAs is one of the core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC uses L-threonine, bicarbonate, and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC moiety is transferred to N6 atom of tRNA A37 to generate t⁶A by TsaD with the support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t⁶A biosynthesis using TsaC, TsaD, TsaB, and TsaE from Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD₂B₂ tetramer. Our TsaD₂B₂-TsaE-tRNA model coupled with functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1-α2 of TsaD. A. aeolicus TsaDB possesses a basal t⁶A catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that the binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7, and α8 of TsaD and C-terminal tail of TsaB, leading to the release of tRNA t⁶A and AMP. ATP-mediated binding of TsaE to TsaDB resets a t⁶A active conformation of TsaDB. Dimerization of TsaDB enhances thermostability and promotes t⁶A catalysis of TsaD₂B₂-tRNA, of which GC base pairs in anticodon stem are needed for the correct folding of thermophilic tRNA at higher temperatures.

Keywords: ATP hydrolysis; Aquifex aeolicus; GC base pairs content; TsaD(2)B(2) tetramer; TsaD–TsaB–TsaE–tRNA assembly; crystal structure; enzymatic reconstitution; oligomerization; tRNA t(6)A; thermostability.

Figure 1

Functional and structural characterization of TsaC, TsaD, TsaB, and TsaE fromAquifexaeolicus (Aa). A, reaction scheme of tRNA t⁶A biosynthesis by TsaC, TsaD, TsaB, and TsaE from bacteria. In the first step, TsaC protein uses L-threonine, HCO₃^-, and ATP to generate an unstable intermediate threonylcarbamoyladenylate (TC-AMP); in the second step, TsaDB complex transfers the TC-moiety from TC-AMP onto the N6 atom of tRNA A37, leading to tRNA t⁶A. The t⁶A-catalytic cycle of TsaDB is stimulated by TsaE at cost of ATP consumption. B, size-exclusion chromatography (SEC) profiles and SDS–PAGE analysis of A. aeolicus TsaD₂B₂, TsaC, TsaE, and Thermotoga maritima (Tm) TsaD₂B₂. C, crystal structure of A. aeolicus TsaD₂B₂ tetramer. TsaD and TsaB are shown in red and green, respectively. One TsaD–TsaB–Fe²⁺ protomer is cartooned as helices and the other TsaD–TsaB–ADP–Fe²⁺ protomer is cartooned as cylindrical helices. 2F_o–F_c electron densities are contoured at 1.0 σ for ADP and Fe²⁺ in TsaD and for H¹¹¹–H¹¹⁵–D²⁹⁶ motif and CSO¹⁰ of TsaD. D, structural juxtaposition of A. aeolicus TsaD–ADP–Fe²⁺ (colored in red) and E. coli (Ec) TsaD–BK951–Zn²⁺ (PDB: 6Z81, colored in cyan). The close-up view shows the binding orientations of ADP or TC-AMP (BK951) in the catalytic sites of TsaD. The dashed line denotes the distance from CSO¹⁰ to the threonyl-moiety of TC-AMP. E, t⁶A-modification efficiencies of 5 μM A.aeolicus TsaD₂B₂E₂ and A. aeolicus TsaD^C10A₂B₂E₂ in combination with 5 μM TsaC toward 60 μM in vitro transcribed (IVT) A. aeolicus tRNA^Lys_UUU, and t⁶A-modification efficiencies of 5 μM EDTA-dialyzed TsaD₂B₂ toward 60 μM metal-deprived IVT A. aeolicus tRNA^Lys_UUU in assays supplemented with 30 μM isolated TC-AMP and 100 μM MgCl₂, FeCl₂ or ZnCl₂. Error bars represent standard deviations from triplicate measurements. F, structural juxtaposition of A. aeolicus TsaB and E. coli TsaB (PDB: 4YDU) manifests conserved N-terminal subdomains but varied C-terminal subdomains. The specific insertion segment (residues 143–162) of E. coli TsaB is highlighted in red. G, topological diagrams of A. aeolicus TsaB (green) and E. coli TsaB (gray). The secondary structures (β₉α₆β₁₀) of the inserted segment of E. coli TsaB are shown in red. IVT, in vitro transcribed; PDB, Protein Data Bank; t⁶A, N⁶-threonylcarbamoyladenosine.

Figure 2

Structural and functional characterization of TsaD₂B₂tetramer and TsaDB dimer. A, structural juxtaposition of AaTsaD₂B₂ and TmTsaD₂B₂ (PDB: 6N9A) is labeled and shown in different colors. One TsaD–TsaB protomer is cartooned as helices and the other TsaD–TsaB protomer is cartooned as cylindrical helices. The close-up views show the interacting residues of AaTsaB–TsaB and TmTsaB–TsaB. B, SEC profiles of TsaDB dimers from Aquifex aeolicus, Thermotoga maritima, and Escherichia coli. C, SEC analysis of the interaction between A. aeolicus TsaDB and TsaE. Briefly, 400 μg TsaDB and 450 μg TsaE (1:4 in molar ratio) was applied to Superdex 75 10/300 GL column (GE HealthCare) and the inset shows SDS–PAGE analysis of proteins in fractions corresponding to the major peaks. D, microscale thermophoresis (MST) measurements of the interaction between 10 nM 5′ 6-FAM-tRNA^Lys_UUU and 195 nM–100 μM TsaD₂B₂ tetramer or TsaDB dimer from A. aeolicus, T. maritima or E. coli. E, comparison of tRNA t⁶A modification efficiencies in complete assays using 5 μM TsaD₂B₂ or TsaD₂B₂E₂ from A. aeolicus and T. maritima, 10 μM TsaDB or TsaDBE from A. aeolicus and T. maritima, and 5 μM TsaDB or TsaDBE from E. coli. Error bars represent standard deviations from triplicate measurements. t⁶A, N⁶-threonylcarbamoyladenosine; SEC, size-exclusion chromatography.

Figure 3

Structural model of Aquifexaeolicus TsaD₂B₂in complex with TsaE or tRNA and functional validations. A, a structural model for A. aeolicus TsaD₂B₂–TsaE–tRNA complex. tRNA is bound to one TsaDB protomer and TsaE is bound to the other TsaDB protomer. B, a close view of the assemble of A. aeolicus TsaE onto TsaDB. C, a close view of the binding of anticodon stem loop of tRNA in the catalytic center of TsaD. The helical hairpin α1–α2 of A. aeolicus TsaD are shown in yellow; the equivalent α1 of Thermotoga maritima TsaD and C-terminal α7 of T. maritima TsaB are shown in gray and indicated with stars. The other parts of T. maritima TsaDB are omitted for clarity. D, local sequence alignment of residues 32 to 42 of TsaDs from A. aeolicus, T. maritima, and E. coli, and the schematic representation of A. aeolicus TsaD^Δ34–38 mutant. E, local sequence alignment of the C-terminal tail of TsaBs from A. aeolicus, T. maritima, and E. coli, and the schematic representation of A. aeolicus TsaB^mut mutant. F–H, ITC analysis of the interaction between A. aeolicus TsaD₂B₂ (F), TsaD^Δ34–38₂B₂ (G) or TsaD₂B^mut₂ (H) and TsaE. Representative plots from an ITC experiment are shown with raw data in the upper panel and curve fit in the lower panel. I, MST measurements of the interaction between 10 nM 5′ 6-FAM-tRNA^Lys_UUU and 195 nM–100 μM TsaD₂B₂, TsaD^Δ34–38₂B₂ or TsaD₂B^mut₂. J, quantification of tRNA t⁶A modification efficiencies of 5 μM A.aeolicus TsaD₂B₂E₂, TsaD^Δ34–38₂B₂E₂ or TsaD₂B^mut₂E₂ toward 60 μM IVT tRNA^Lys_UUU. Error bars represent standard deviations from triplicate measurements. K, EMSA analysis of the competition binding of TsaE and tRNA to TsaD₂B^mut₂. TsaE at indicated concentration was added to 5 μM AaTsaD₂B^mut₂ that was preincubated with 1 μM 5′ 6-FAM-tRNA^Lys_UUU. The upper panel shows the presence of 5′ 6-FAM-tRNA^Lys_UUU and bottom panel shows the presence of AaTsaD₂B^mut₂E₂ and unbound TsaE. IVT, in vitro transcribed; 6-FAM, 6-carboxyfluorescein; ITC, isothermal titration calorimetry; MST, microscale thermophoresis; t⁶A, N⁶-threonylcarbamoyladenosine; EMSA, electrophoresis mobility shift assay.

Figure 4

Analysis of ATPase activity of TsaE in the presence of TsaDB and tRNA. A, initial velocities of 4 mM ATP to ADP hydrolysis by 10 μM A. aeolicus TsaE, 5 μM A.aeolicus TsaD₂B₂E₂, 5 μM E.coli TsaE, 5 μM E.coli TsaDBE, 10 μM T. maritima TsaE, or 5 μM T.maritima TsaD₂B₂E₂. Error bars represent standard deviations from triplicate measurements. B, steady-state kinetics analysis of the ATPase activity of 10 μM A.aeolicus TsaE in the presence of 5 μM A.aeolicus TsaD₂B₂ or mutants and 10 μM IVT tRNA^Lys_UUU. Kinetic parameters are summarized in Table 2. Error bars represent standard deviations from triplicate measurements. IVT, in vitro transcribed

Figure 5

Thermostability analysis of tRNA t⁶A biosynthetic systems from Aquifex aeolicus, Thermotoga maritima and Escherichia coli.A, melting temperature (T_m) values of TsaD₂B₂ or TsaDB from A. aeolicus, T. maritima, or E. coli determined by thermal shift assay. Error bars represent standard deviations from triplicate measurements. B, LC–MS chromatograms of TC-AMP formation in assays that contained 5 μM AaTsaC, 1 mM ATP, 4 mM L-threonine, and 10 mM NaHCO₃ at 25 °C or 75 °C. C, quantification of tRNA t⁶A modification efficiency by 5 μM TsaD₂B₂E₂ or 10 μM TsaDBE from A. aeolicus or T. maritima toward 60 μM cognate IVT tRNA^Lys_UUU at 55 °C or 75 °C. For the assay with 60 μM E.coli tRNA^Lys_UUU, 5 μM A.aeolicus TsaD₂B₂E₂ was used. Error bars represent standard deviations from triplicate measurements. D, sequence alignment of tRNA^Lys_UUU from A. aeolicus, T. maritima, and E. coli. Amino acid acceptor arms, D–stems, anticodon stems, and TψC stems are shown in gray, pink, green, and blue, respectively. GC pairs and anticodons are highlighted in black and red, respectively. E, schematic representation of the GC base pairs mutations of E. coli tRNA^Lys_UUU variant 1 and variant 2. F, T_m values of A. aeolicus tRNA^Lys_UUU, T. maritima tRNA^Lys_UUU, E. coli tRNA^Lys_UUU, EctRNA^Lys_UUU variant 1 and variant 2, which were determined by circular dichroism spectra analysis. G, LC–MS chromatograms of tRNA t⁶A formation by AaTsaC and AaTsaD₂B₂E₂ in assay using E. coli tRNA^Lys_UUU, variant 1 and variant 2 at 55 °C or 75 °C. IVT, in vitro transcribed; t⁶A, N⁶-threonylcarbamoyladenosine