Probing the stereospecificity of tyrosyl- and glutaminyl-tRNA synthetase with molecular dynamics

The stereospecificity of aminoacyl-tRNA synthetases helps exclude D-amino acids from protein synthesis and could perhaps be engineered to allow controlled D-amino acylation of tRNA. We use molecular dynamics simulations to probe the stereospecificity of the class I tyrosyl- and glutaminyl-tRNA synthetases (TyrRS, GlnRS), including wildtype enzymes and three point mutants suggested by three different protein design methods. L/D binding free energy differences are obtained by alchemically and reversibly transforming the ligand from L to D in simulations of the protein–ligand complex. The D81Q mutation in Escherichia coli TyrRS is homologous to the D81R mutant shown earlier to have inverted stereospecificity. D81Q is predicted to lead to a rotated ligand backbone and an increased, not a decreased L-Tyr preference. The E36Q mutation in Methanococcus jannaschii TyrRS has a predicted L/D binding free energy difference ΔΔG of just 0.5 ± 0.9 kcal/mol, compared to 3.1 ± 0.8 kcal/mol for the wildtype enzyme (favoring L-Tyr). The ligand ammonium position is preserved in the D-Tyr complex, while the carboxylate is shifted. Wildtype GlnRS has a similar preference for L-glutaminyl adenylate; the R260Q mutant has an increased preference, even though Arg260 makes a large contribution to the wildtype ΔΔG value.