Protein Engineering on Leucyl-tRNA Synthetase (LeuRS)

    Each of the twenty tRNA systhetases (RS) is responsible for covalently linking a particular amino acid to its cognate tRNA. Through a two-step reaction, the enzyme forms an aminoacyl adenylate (AMP) and then transfers the activated amino acid to the 3' end of the tRNA. The 'charged' tRNA shuttles the amino acid to the directing protein systhesis.  Although the reaction is highly specific, some RS can activate and aminoacylate incorrect amino acids which structurally overlap with the cognate amino acid. High fidelity of these RS is maintained via an extra domain of about 200-300 amino acids containing  a hydrolytic active site that proofreads and edits misactivated or mischarged aminoacids.

    Our goal is 'Protein Engineering' or 'Protein Design' by controling of tRNA synthetase.  By mutation of some proper residue(s),  we expect to be able to control the amino-acid recognition ability(or characteristics)  of  a tRNA synthetase.  In order to achieve this goal, we chose the leucyl tRNA synthetase system as a prove by the relationship with cowork group (Martinis Group, University of Houston).

    Unfortunately, the 3D structure of E. coli LeuRS is not known yet. Therefore we built the homology modeling structure of E. coli LeuRS using the X-ray structure of T. thermophilus LeuRS (K.W. Lee et al. Proteins 54(4) 693-704 (2004)).

Fig.1. X-ray structures of T. Thermophilus LeuRS (A) and homology modeled structure of E. Coli LeuRS(B)
    From the refine 3D structure, the binding mode of the ligand leucine and LeuRS was studied by the DOCKING simulation. Based on the information from DOCKING result, the mutation residue will be decided and then at the same time, many molecular dynamics (MD) simulations were followed to investigate the interaction between the ligand and the protein.


Fig.2 Binding of Leucyl-AMP to the editing active site of E. coli LeuRS