Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.

Markus Englert,Yane-Shih Wang,Daniel Eiler,Akiyoshi Nakamura,Dieter Söll,Li-Tao Guo

doi:10.1093/nar/gkv1255

Abstract

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

Highlights

Trp is one of the 20 canonical amino acids, which plays unique roles in maintaining protein folds and promoting protein–protein and protein–ligand interactions, because of the various types of non-covalent interactions formed by its large hydrophobic yet polar aromatic side chain [1,2,3,4,5]
New pyrrolysyl-tRNA synthetase (PylRS) variants were selected from a mutation library of Methanosarcina mazei PylRS containing three types of combinations of random mutations: (i) a previously described group with mutations of Leu305, Asn346 and Cys348 [20]; (ii) a group with mutations of Asn346, Cys348 and Tyr306 and (iii) a group with mutations of Asn346, Cys348, Leu309, with indicated residues randomized as NNK in all three sub-libraries
The enzymes were screened for incorporation of the noncanonical amino acids (ncAAs) 3-I-Phe and selected via three rounds of the standard positive-negative-positive selection procedure in E. coli TOP10 cells [20,26]

Summary

Introduction

Trp is one of the 20 canonical amino acids, which plays unique roles in maintaining protein folds and promoting protein–protein and protein–ligand interactions, because of the various types of non-covalent interactions formed by its large hydrophobic yet polar aromatic side chain [1,2,3,4,5]. For Bta two systems have been used to date: a mutated yeast phenylalanyl-tRNA synthetasetRNAPheCUA pair [13] and an optimized S. cerevisiae tryptophanyltRNA synthetasetRNATrpCUA [14,15]

Methods

Results

Conclusion