An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity

Abstract

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.

Figure 1

GFP fluorescence assay for pCNF-RS dependent incorporation of unnatural amino acids into GFP_Y151X. A 96-well plate contains different non-native amino acids (1 mM, see supporting information) incubated with BL-21(DE3) cells harboring the GFP_Y151X reporter and the pEVOL-pCNF in 2YT media. Fluorescence was measured after 14 h at 30 °C of expression and corrected for background by a non-induced culture.

Figure 2

Unnatural amino acids screened for incorporation by pCNF-RS

Figure 3

A) Overlay of the key active site residues for substrate bound pAcF-RS·pAcF (cyan; PDB 1ZH6) and pCNF-RS·pCNF (green) complexes. The pAcF (blue) and pCNF (gray) substrates are show at the center of the structure. B) Overlay of the substrate bound pNapA-RS·NapA (magenta, PDB 1ZH0) and pCNF-RS·pCNF (green) complexes. The NapA (magenta)and pCNF (green) substrates are showat the bottom of the structure.

Figure 4

Structural analysis of 3 key residues (32, 65, and 158) for amino acid recognition in various synthetases. A) Wild-type tyrosyl RS (green, PDB 1J1U) modeled with pCNF. B) pCNF-RS (cyan) modeled with pCNF. C) pAcF-RS (magenta, PDB 1ZH6) modeled with pCNF. D) NapA-RS (orange, PDB 1ZH0) modeled with pCNF. E) pCNF-RS (cyan) modeled with pAcF. F) pAcF-RS (magenta, PDB 1ZH6) modeled with pAcF.

Figure 5

Backbone pertubations in various aminoacyl-tRNA synthetases. A) Overlay of the helices containing mutated residues 158 and 159 in NapA-RS (orange; PDB 1ZH0), WT-TyrRS (green; PDB 1J1U), pAcF-RS (magenta; PDB 1ZH6) and pCNF-RS (cyan). The termination of the helix which accommodates large amino acids is especially apparent in NapA-RS B) Overlay of the helices containing mutated residues 108 and 109 in WT-TyrRS (green; PDB 1J1U), pAcF-RS (magenta; PDB 1ZH6) and pCNF-RS (cyan). pCNF = cyan, pAcF = magenta, NapA = orange.