title: Redesigning the stereospecificity of tyrosyl-tRNA synthetase.
abstract: D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Simonson
authors.affiliation: Department of Biology, Ecole Normale Supérieure, Paris, France.
authors.forename: Shixin
authors.initials: S
authors.lastname: Ye-Lehmann
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Palmai
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Najette
authors.initials: N
authors.lastname: Amara
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Wydau-Dematteis
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Erwan
authors.initials: E
authors.lastname: Bigan
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Karen
authors.initials: K
authors.lastname: Druart
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Clara
authors.initials: C
authors.lastname: Moch
authors.affiliation: Department of Biology, Laboratoire De Biochimie (CNRS UMR7654), Ecole Polytechnique, Palaiseau, 91128, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Plateau
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering; D013237:Stereoisomerism; D013816:Thermodynamics; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D014448:Tyrosine-tRNA Ligase
keywords: computer simulations; free energy; genetic code; protein design