Strictly conserved lysine of prolyl-tRNA Synthetase editing domain facilitates binding and positioning of misacylated tRNA(Pro.)

Abstract

To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA(Pro). This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2'-OH of misacylated tRNA(Pro) and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNA(Pro) deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA(Pro) hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA(Pro) and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA(Pro) hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.

Figure 1

Ribbon representation of the three-dimensional structural model of the monomeric form of Ec ProRS. The homology model was derived from the X-ray crystal structure of Ef ProRS (PDB code: 2J3L). The catalytically important residues and the GXXXP motif of Ec ProRS are shown by licorice representation; the Ef ProRS residues are shown in parentheses.

Scheme 1. Scheme for the pK_a Calculation of Lys279 using Kirkwood’s Thermodynamic Integration Method

Scheme 2. A Thermodynamic Diagram for the Calculation of Gibbs Free Energy of Binding of the 5′-CCA-Ala Substrate to the INS Domain

Figure 2

A plot of the partial derivative of Gibbs free energy with respect to the coupling parameter λ, (∂G/∂λ), as a function of the coupling parameter, λ.

Figure 3

(a) The highly conserved K279 and the surrounding polar residues are shown in the editing active site of Ef ProRS. (b) Sequence alignment of the relevant portion of INS domain from Ec and Ef ProRS.

Figure 4

Change in the QM/MM interaction energy due to charge removal of neighboring polar residues of K279, averaged over 100 conformations.

Figure 5

Altered conformation of the 5′-CCA-Ala bound to the INS double mutant (b) compared to WT Ef ProRS (a). The protein segment is shown in new cartoon representation, whereas the 5′-CCA-Ala substrate is represented in licorice.

Figure 6

Initial rates of aminoacylation of tRNA^Pro with proline by WT and the five variants of Ec ProRS. For clarity, the results are presented in two panels, (a) and (b). The assays were performed at room temperature with 0.5 μM tRNA^Pro and 100 nM Ec ProRS. Linear fits of the data are shown.

Figure 7

Relative pretransfer editing activity of WT and mutant Ec ProRS. The assay was performed at 37 °C using 4 μM ProRS and 500 mM alanine. Results are reported as percent activity relative to WT, which was set to 100%.

Figure 8

Deacylation of Ala-tRNA^Pro by WT and mutant Ec ProRS. The assays were performed at room temperature with 1 μM G1:C72/U70 [¹⁴C]Ala-tRNA^Pro and 0.5 μM Ec ProRS.