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.
A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl-tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l-tyrosine (l-Tyr), compared to the analogs d-Tyr, p-acetyl-, and p-azido-phenylalanine (ac-Phe, az-Phe). We simulate l- and d-Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous "MD/GBSA" procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l-Tyr, ac- and az-Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l-Tyr or d-Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l-Tyr is the ligand and a d-Tyr specific mutant when d-Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln.
3-Phosphogycerate kinase (PGK) is a two domain enzyme, with a binding site of the 1,3-bisphosphoglycerate on the N-domain and of the ADP on the C-domain. To transfer a phosphate group the enzyme has to undergo a hinge bending motion from open to closed conformation to bring the substrates to close proximity. Molecular dynamics simulation was used to elucidate the effect of ligand binding onto the domain motions of this enzyme. The simulation results of the apo form indicate a hinge bending motion in the ns timescale while the time period of the hinge bending motion of the complex form is clearly over the 20 ns simulation time. The apo form exhibits several hinge points that contribute to the hinge bending motion while upon binding the ligands, the hinge bending becomes strictly restrained with one dominant hinge point in the vicinity of the substrates. At the same time, ligand binding results in an enhanced correlation of internal domain motions.
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