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.

Protein:Ligand binding free energies: A stringent test for computational protein design

“…The new values are much smaller (Table ), ranging from +1.8 kcal/mol (wildtype; l ‐Tyr preference) to −2.5 kcal/mol (KYEQ; d ‐Tyr preference). For DYQQ and RYQQ, they are reasonably consistent with the results from rigorous, alchemical MD free energy simulations, although for NYQQ, the MC value is smaller than the alchemical result. Importantly, the ordering of the seven sequences is maintained when we change N min ( d ‐Tyr).…”

“…The MC‐min results show rough agreement (Table ) with rigorous alchemical MD free energy simulations, available for DYQQ, RYQQ, and NYQQ . For DYQQ and RYQQ, the results differ by 0.7 and 1.6 kcal/mol, which is significant but no larger than the statistical noise.…”

“…We consider first the binding of l ‐Tyr/ d ‐Tyr to wildtype TyrRS and six mutants. The proteins are named according to the four positions allowed to mutate in earlier design work: residues 81, 175, 179, and 201, whose native types are Asp, Tyr, Gln, and Gln, so that we call the wildtype protein “DYQQ.” The locations of the designed positions in the binding pocket are shown in Figure . The six mutants studied here are RYQQ, KYQQ, KYEQ, KYED, HYED, and NYQQ (Table ).…”

“…Whereas most aminoacyl‐tRNA synthetases aaRSs have a strong preference for their l ‐amino acid substrate, TyrRS has a detectable, natural activity for d ‐tyrosine . We recently characterized several candidate mutations that were designed to make TyrRS specific for d ‐Tyr . Such a modified TyrRS could potentially be used to insert d ‐Tyr into proteins in vivo .…”

“…With the native, l ‐Tyr ligand, we recovered the native amino acid types a large fraction of the time at positions 81 and 179, while the design of position 201 yielded His instead of the native Gln. With the non‐native, d ‐Tyr ligand, we predominantly recovered a single‐site D81R mutant that has been shown experimentally to be active and to have an inverted stereospecificity, with a preference for the d ‐Tyr substrate …”