Importance of single molecular determinants in the fidelity of expanded genetic codes

Abstract

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

Fig. 1.

Molecular interactions between hydrogen-bonding side chains from Bst tyrosyl tRNA synthetase with bound Tyr-AMP. Bst residues Tyr34 and Asp174 map onto Mj Tyr32 and Asp158, respectively. (Reproduced with permission from ref.  (Copyright 1985, Nature Publishing Group.)

Fig. 2.

Analysis of the revertants (A) and the original AARS mutants (B). Tyr32 was reintroduced into the three TyrRS variants indicated (the other mutations to these variants are listed in Table S1). The lanes are labeled (+) or (-) that designates whether the corresponding nonnatural amino acid was added to the culture. The SDS-PAGE analysis shown was performed after Ni²⁺-affinity purification of full-length T4 lysozyme purified by the C-terminal hexahistidine tag to remove the truncated material and simplify the analysis. The lane furthest left corresponds to a commercial protein ladder and a molecular weight band of 20 kDa.

Fig. 3.

Alignment of TrpRSs from B. stereothermophilus (magenta) and a V144P TrpRS from B. subtilis (green and yellow; PDB ID 3PRH). Bound substrate Trp (shown as space-filling model) is from Carter’s B. stereothermophilus structure (PDB ID 3FHJ1). Two Asp residues, one from each RS, are also shown near the indole nitrogen. These Asp residues are the determinants for amino acid specificity. The effect of the ProPro sequence on the B. subtilis secondary structure is shown in yellow and occurs where the nucleoside would normally reside.

Fig. 4.

B. stereothermophilus active site with Val143Pro mutation to model the effects of the Bs Val144Pro (5-OH indole ring in green; L-Trp indole ring shown in orange). Highly similar results were observed for the WT Bst TrpRS.

Fig. 5.

Complementation of E. coli trpS4040 by (Left) 0.05 μM Trp, and (Right) 5 μM 5-OH Trp. Figure shows growth after 48 h at 37 °C. (1 = pET151 Bs TrpRS (wild type)/pGP1-2; 2 = pET151 Bs TrpRS (V144P)/pGP1-2; 3 = pET151ϵ (control)/pGP1-2; M9 minimal media + 50 μg/mL ampicillin and 25 μg/mL kanamycin). The results on plates were repeated three times.

Fig. 6.

Isothermal calorimetry results with tryptophan and 5-OH Trp ligands. (A) Bs WT TrpRS with L-Trp; (B) Bs WT TrpRS with 5-OH Trp; (C) Bs TrpRS V144P variant with L-Trp; (D) Bs TrpRS V144P variant with 5-OH Trp.