Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain

Abstract

High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Figure 1.

NMR Chemical shift perturbations (CSPs) implicate Lys50 and Arg80 of Cc ProXp-ala in tRNA^Pro recognition. (A) Secondary structure of microhelix^Pro with the WT C1:G72 base pair (green) and the G1:C72 variant (blue). (B) Overlaid ¹H-¹⁵N HSQC spectra of [U-¹⁵N]-ProXp-ala (black) alone and in the presence of WT- (green) or G1:C72-microhelix^Pro (blue). Perturbations of select residues are labeled and indicated by arrows. (C) Secondary structure of Cc ProXp-ala (top) and magnitude of per residue CSPs induced by WT- (green) and G1:C72-microhelix^Pro (blue) (bottom). (D) Spectral expansions highlighting CSPs for Lys50 and Arg80 (left) and Leu47 (right). (E) CSP differences between WT- and G1:C72-microhelix^Pro-bound states mapped with a linear color gradient from white (0 ppm) to dark green (0.25 ppm) on a cartoon representation of the Cc ProXp-ala crystal structure (PDB: 5VXB). Unassigned residues are grey, and Lys50 and Arg80 are highlighted in blue.

Figure 2.

Arg80 and Lys50 are critical for both RNA binding and substrate deacylation. (A) Analytical ultracentrifugation sedimentation velocity analysis of 1 μM WT microhelix^Pro alone (dashed line), and in the presence of 9.6 μM WT (black), R80A (green) or K50A (blue) ProXp-ala. Inset table shows s_w values obtained from integrated c(s) distributions. (B) Single-turnover deacylation assays of 0.1 μM Ala-tRNA^Pro by 0.75 μM WT (black), R80A (green) and K50A (blue) ProXp-ala. Inset shows an expanded view of the WT and K50A ProXp-ala data. Lines represent single- (R80A) and double- (K50A and WT) exponential fits of the data. Error bars are the standard deviation of three replicates. All deacylation analyses were performed after correcting for nonenzymatic buffer hydrolysis at each timepoint.

Figure 3.

Single-turnover deacylation assays show that K50A and R80A ProXp-ala variants display reduced sensitivity to tRNA^Pro acceptor stem mutants compared to WT ProXp-ala. (A) Single-turnover deacylation assays performed with WT (closed circles) or R80A ProXp-ala (open circles), using either WT (black), G1:C72- (orange), or C73- (green) Ala-tRNA^Pro. Lines represent fits to a double-exponential equation in the case of WT ProXp-ala and WT or A73C tRNA^Pro; the other lines represent single-exponential fits of the data. Error bars are the standard deviation of three replicates. All deacylation analyses were performed after correcting for nonenzymatic buffer hydrolysis at each timepoint. (B) Summary of k_obs values (min⁻¹) for WT, K50A, R80A and R80N ProXp-ala, and WT (gray), G1:C72-(orange) and C73-(green) tRNA^Pro. In cases where double-exponential fits were used (WT ProXp-ala and WT or A73C tRNA^Pro; K50A ProXp-ala and WT tRNA^Pro), k_obs values were obtained from the fast phase, which represented at least 80% of the amplitude (see Supplementary Table 3).

Figure 4.

Docking model of the truncated microhelix^Pro-ProXp-ala complex shows inferred interactions between Arg80 and Lys50 side chains and the C1:G72 base pair major groove. Overview (A) and zoomed in view (B). Distances in Å between Lys50 Nζ and Arg80 Nη atoms and C1 N4 and G72 N7 and O6 are indicated by dashed lines.

Figure 5.

ProXp-ala binding to tRNA^Pro is sensitive to changes in A76. (A) Left: schematic of RNA duplex variants used in EMSA assays where one strand is fluorescently labeled at the 3′ end. Right: Results of EMSA binding assays plotting fraction of bound RNA as a function of ProXp-ala concentration; A76 (WT, green), ΔA76 (blue) and 3′pΔA76 (orange). Lines are Hill equation fits to the data; error bars are standard deviation of three replicates. (B) Overlaid ¹H-¹⁵N HSQC spectra for the amide nitrogens of Glu30 (top) and Lys71 (bottom) illustrate decreased CSPs upon A76 mutations: Free [U-¹⁵N]-ProXp-ala (black), [U-¹⁵N]-ProXp-ala in the presence of the ΔA76-microhelix^Pro (blue), 3′pΔA76-microhelix^Pro (orange), or WT (A76) microhelix^Pro (green). The arrows indicate the CSPs observed upon WT microhelix^Pro binding to free ProXp-ala.

Figure 6.

CSPs show parallel ProXp-ala-tRNA^Pro binding deficiencies upon deletion of A76 and mutation of K45. (A) Schematic of minihelix^Pro, which is indistinguishable from microhelix^Pro in terms of its binding to ProXp-ala (Supplementary Fig. 4). (B) Secondary structure of Cc ProXp-ala (top) and summary of per residue CSPs induced by minihelix^Pro binding to WT (green) and K45A (orange) ProXp-ala. (C) Spectral expansions of Gly84 (top), Lys71 (middle) and Leu47 (bottom) from overlaid ¹H–¹⁵N HSQC spectra of WT ProXp-ala (left and middle spectra) free (black), and in the presence of microhelix^Pro (green) or ΔA76-microhelix^Pro (blue), and of K45A ProXp-ala (right spectra) free (black), and in the presence of minihelix^Pro-bound (orange).

Figure 7.

The 2′ OH of A76 is important for deacylation of Ala-tRNA^Pro by ProXp-ala. Deacylation assay with 0.75 μM ProXp-ala and 0.1 μM Ala-A76-tRNA^Pro (dark grey) or Ala-dA76-tRNA^Pro (light grey). Assays were performed in triplicate with the mean value of aminoacyl-tRNA remaining after 30 min indicated by the top of the bar.