Reassignment of sense codons: Designing and docking of proline analogs for Escherichia coli prolyl-tRNA synthetase to expand the genetic code

Amino acyl-tRNA synthetases (AARSs) play a vital role in protein synthesis by catalyzing the aminoacylation of tRNA with its cognate amino acid. More recently, the endogenous AARS has been reported to recognize the close structural analogs of its cognate amino acid and helps in the in vitro and in vivo incorporation of analogs into recombinant proteins. By exploiting this substrate promiscuity, a number of non-canonical amino acids were successfully incorporated into the recombinant proteins. However, the incorporation efficiency varies with the different structural analogs depending on their reactivity towards the tRNA synthetases, which is due to the interaction and accommodation in the active site. Here, to analyze the incorporation efficiency of different proline analogs and to predict the active site residues responsible for the recognition, we carried out molecular docking study with the modeled Escherichia coli prolyl-tRNA synthetase (EcProRS). We also mapped the binding mode for the reported, virtually generated proline analogs and compared it with the reported crystal structure. The reactivity of the reported analogs was correlated with the biochemical data with respect to their interaction and orientation in the active site, which demonstrates the role of active site residues for the recognition of proline analogs and some new substrates such as chloro, bromo and iodoproline for EcProRS. We also rationally designed a EcProRS mutant for desired proline analog and validated by docking simulation with 3D model structure.

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► We performed Docking study to predict substrate selectivity of Prolyl tRNA synthetase.
► Key interactions and residues responsible for proline analog binding was identified.
► To develop novel substrate for ProRS, optimal side chain substitution position of prolyl ring was predicted.