Isoleucyl transfer RNA (tRNA) synthetase (IleRS) joins Ile to tRNAIle at its synthetic active site and hydrolyzes incorrectly acylated amino acids at its editing active site.
The 2.2 angstrom resolution crystal structure of Staphylococcus aureus IleRS complexed with tRNAIle and Mupirocin shows the acceptor strand of the tRNAIle in the continuously stacked, A-form conformation with the 3′ terminal nucleotide in the editing active site.
We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNAPro and position it in the editing active site.
Thus, the combined computational and experimental results support the conclusion that K279 is involved in binding and positioning the misacylated tRNAPro into the post-transfer editing active site.
A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.
Previous studies of Escherichia coli (Ec) ProRS have demonstrated that the aminoacylation domain is the site of pretransfer editing, whereas an insertion domain located between the class II synthetase conserved motifs 2 and 3 (INS, ∼180 amino acids) is the post-transfer editing active site that hydrolyzes Ala-tRNAPro.
On the basis of both experimental and computational results, it has been speculated that the role of this strictly conserved basic residue in prokaryotic-like ProRSs and related editing domains is to bind the misacylated tRNA substrate and position it in the editing active site.
On the basis of previous experimental, substrate docking, and MD simulation results, we hypothesized that the role of the conserved lysine may be to anchor the phosphate backbone of the acceptor stem of misacylated tRNAPro and position it into the editing active site.
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The extent by which tRNA stimulates editing also dropped from 7 to 1.5 (Table 1), indicating that tRNA enhancement of the Y59T editing activity originates from the active post-transfer pathway.
