Prolyl-tRNA synthetases (ProRSs) are multi-domain proteins that catalyze covalent attachment of proline to the 3�-end of the tRNAPro. ProRSs from all three kingdoms of life are known to misactivate alanine and form mischarged tRNAPro (1, 2). To maintain high fidelity in protein synthesis, some ProRSs have acquired editing mechanisms. The insertion domain (INS, ~180 amino acids) of Escherichia coli (Ec) ProRS is the post-transfer editing active site that hydrolyzes specifically mischarged alanyl-tRNAPro(1, 2).  Experimental studies have demonstrated that the INS domain of Ec ProRS also has a significant impact on amino acid binding and activation (3). To identify what exact role the INS domains (editing domain) plays in amino acid activation, the dynamic coupling between the editing domain and various structural elements of the catalytic domain of Ec ProRS was studied using molecular dynamics simulations. Moreover, statistical-thermal coupling analysis was performed to identify residue-residue interaction networks that facilitate coupled-domain dynamics in Ec ProRS. Site-directed mutagenesis and kinetic studies are being carried out to explore the impact of mutations (of residues within the interaction networks) on the enzyme activity. Herein, we will report the computational and experimental results that demonstrate the role of the editing domain in maintaining the intrinsic protein dynamics and stabilizing the catalytically important proline-binding loop.  References: 1) Beuning, P. J., and Musier-Forsyth, K. (2000) Proc. Natl. Acad. Sci. U .S. A. 97, 8916-8920.2. Beuning, P. J., and Musier-Forsyth, K. (2001) J. Biol. Chem .276, 30779-30785.3) Hati, S., Ziervogel, B., Sternjohn, J., Wong, F. C., Nagan, M. C., Rosen, A. E., Siliciano, P. G., Chihade, J. W., and Musier-Forsyth, K. (2006) J. Biol. Chem. 281, 27862-27872.

Computational and experimental studies to unravel the role of editing domain of bacterial prolyl-tRNA synthetases in amino acid activation

243rd National Meeting (2012)

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