Structural basis for aminoacylation of cellular modified tRNALys3 by human lysyl-tRNA synthetase

Abstract

The average eukaryotic transfer ribonucleic acid (tRNA) contains 13 post-transcriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully modified cellular tRNALys3 by human lysyl-tRNA synthetase (h-LysRS). The tRNALys3 anticodon loop modifications S34 (mcm5s2U) and R37 (ms2t6A) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNALys3 are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNALys3 aminoacylation are structurally ordered; docking of the 3'-CCA end in the active site cannot proceed until the lysyl-adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS-tRNALys3 complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium toward the 3'-CCA end "docked" conformation and allosterically increases h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.

Graphical Abstract

Figure 1.

Structure of h-LysRS bound to modified cellular tRNA^Lys3 and AIMP2. (A) A schematic representation of the MSC. h-LysRS dimer is anchored to the MSC by AIMP2 and is highlighted. (B) Cryo-EM reconstruction of wt h-LysRS bound to modified cellular tRNA^Lys3 and AIMP2 in the presence of AMP and L-lysine. (C) Structural model built for the h-LysRS–tRNA^Lys3–AIMP2 complex based on the cryo-EM reconstruction shown in panel (B). The various domains of h-LysRS are highlighted. (D) Representative modified nucleotides in tRNA^Lys3 are shown with the corresponding cryo-EM map density from the reconstruction shown in panel (B). (E) The N-terminal residues of AIMP2 (teal) secure the dimeric interface between the ABD of one LysRS monomer (purple) and the AAD of the other (coral orange) and are shown in stick representation. (F) Cellular modified tRNA^Lys3 in the h-LysRS bound (gold) and unbound (gray) form is shown. The bottom inset shows the anticodon stem loop of cellular modified tRNA^Lys3 in the unbound form (PDB: 1FIR), highlighting the stacking interactions between the five anticodon loop bases (U33-R37). (G) The anticodon loop of tRNA^Lys3 (U33-R37) bound to h-LysRS is shown along with the corresponding cryo-EM map density. The insets highlight the interactions of the anticodon loop nucleotides (gold) with LysRS (purple).

Figure 2.

tRNA^Lys3 interactions with h-LysRS NTD and active site. (A) A view of the h-LysRS–tRNA^Lys3 complex (h-LysRS in cartoon and tRNA^Lys3 in surface view) showing the NTD helix of LysRS interacting with the elbow region of tRNA^Lys3. The inset highlights the residues at the base of the NTD helix and their contacts in the tRNA^Lys3 T-loop and variable loop. (B) A magnified view showing the N-terminal helix of h-LysRS and its interactions with two dihydrouridine bases of tRNA^Lys3, D20 and D47, of the D-loop and variable loop, respectively. (C) The docked state h-LysRS–tRNA^Lys3 complex is shown, with the insets highlighting key interactions for docking the 3′-CCA end of tRNA^Lys3. LysRS linker residues interact with the D-arm (middle) whereas the acceptor arm is stabilized by a series of ionic interactions (right). (D) A magnified view of the h-LysRS active site is shown with the tRNA^Lys3 3′-CCA end docked in. The corresponding cryo-EM map density is shown in mesh. (E) The interaction map of the h-LysRS–tRNA^Lys3 complex is shown with tRNA^Lys3 portrayed in the conventional clover-leaf representation.

Figure 3.

ATP state governs 3′-CCA end docking of tRNA^Lys3 in the active site. (A) Cryo-EM reconstructions of LysRS bound to IVT tRNA^Lys3 (unmodified) in the presence of L-lysine and either the nonhydrolyzable ATP analog (AMPCPP) (left panel) or the post-hydrolysis ATP analog, AMP (right panel). The AMPCPP bound complex exists solely in the 3′-CCA “undocked” conformation whereas the AMP bound complex can assume the “undocked” and the “docked” conformation. (B) Structural model for the AMPCPP bound “undocked” state of the LysRS–tRNA^Lys3 complex. The right inset shows the interactions of AMPCPP and L-lysine in the active site along with the corresponding cryo-EM map density. (C) The docked state of LysRS–tRNA^Lys3 complex was aligned to the AMPCPP bound “undocked” state. A cross-section view of the LysRS active site is shown highlighting the steric clash between the 3′-CCA end of tRNA^Lys3 and the β- and γ-phosphate of ATP.

Figure 4.

tRNA^Lys3 modifications and AIMP2 enhance h-LysRS activity. (A) Cellular modified tRNA^Lys3 (gold) and IVT unmodified tRNA^Lys3 (sky blue) from their respective docked state structures are shown in cartoon representation with the bases shown as spheres. The nucleotide positions 20 (purple) and 47 (coral) in the modified and unmodified tRNA^Lys3 are highlighted. (B) The docked state structures of LysRS with modified tRNA^Lys3 (gold) and unmodified tRNA^Lys3 (sky blue) were aligned and a magnified view of the linker region interacting with tRNA^Lys3 is shown. Modified tRNA^Lys3 (gold) is shown in surface view and unmodified tRNA^Lys3 is hidden for clarity. LysRS (gold: modified tRNA^Lys3 / sky blue: IVT tRNA^Lys3) is shown in ribbon view. (C) EMSA titrations of h-LysRS binding to unmodified IVT tRNA^Lys3 and modified tRNA^Lys3 were carried out in triplicate. Fraction of tRNA^Lys3 bound by h-LysRS was quantified and the mean is plotted against the protein concentration along with the associated standard error of the mean. The estimated binding affinity (K_d) is denoted along with the associated error from the data fit. (D) The percentage of undocked and docked conformational states of the h-LysRS–tRNA^Lys3 (IVT) complex are shown in bar chart form for the three cryo-EM datasets: h-LysRS–tRNA^Lys3 in presence of AMPCPP and L-lysine, h-LysRS–tRNA^Lys3 in presence of AMP and L-lysine, and h-LysRS–tRNALys3–AIMP2–N36 in presence of AMP and L-lysine. (E) The initial rates of wt and S207D h-LysRS aminoacylation in the presence and absence of AIMP2–N36 peptide, each measured in triplicate. The mean rates are plotted in bar chart form and error bars represent the associated standard deviation. (F) K_m measurement of wt h-LysRS for IVT tRNA^Lys3 in presence and absence of AIMP2 peptide. The normalized aminoacylation rates are plotted against the tRNA^Lys3 concentration. Each data point shown is the average of a triplicate measurement and the error bar corresponds to the associated standard deviation.

Figure 5.

Schematic model for tRNA^Lys3 aminoacylation and the roles of tRNA^Lys3 modifications and the MSC in h-LysRS function. h-LysRS associates with the MSC via the scaffold protein AIMP2. Phosphorylation at S207 residue of h-LysRS prevents association with the MSC. h-LysRS selectively binds tRNA^Lys, and the fidelity of this interaction is imparted by the anticodon loop and the unique tRNA^Lys modifications. tRNA^Lys modifications are also critical in ordering the h-LysRS NTD into an extended helix that docks at the base of the T-loop and interacts along the length of the anticodon arm. The two catalytic steps in tRNA^Lys aminoacylation are structurally ordered by h-LysRS active site. β- and γ-phosphate of the bound ATP creates a steric block and prevents the docking of tRNA^Lys 3′-CCA end. In the ATP-bound state, the h-LysRS–tRNA^Lys complex exists exclusively in the “3′-CCA undocked” state. Once the ATP hydrolysis catalyzed lysyl–adenylate intermediate is formed and the pyrophosphate (PP_i) by-product is released, tRNA^Lys 3′-CCA end can be docked in the active site. In the 3′-CCA docked state, the 3′-A76 nucleotide is unstacked and flipped into the active site, positioning the 3′-hydroxyl of the A76 base for attack on the lysyl–adenylate intermediate. Association with the MSC allosterically drives the equilibrium of the h-LysRS–tRNA^Lys complex from the “undocked” state toward the “docked” state, thereby enhancing the catalytic efficiency of h-LysRS. After the completion of the second step in tRNA^Lys aminoacylation, aminoacylated tRNA^Lys is released along with the byproduct AMP, and the catalytic cycle can begin again.