Functional segregation of a predicted "hinge" site within the β-strand linkers of Escherichia coli leucyl-tRNA synthetase

Some aminoacyl-tRNA synthetases (AARSs) employ an editing mechanism to ensure the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) share a common insertion, called the CP1 domain, which is responsible for clearing misformed products. This discrete domain is connected to the main body of the enzyme via two β-strand tethers. The CP1 hydrolytic editing active site is located ∼30 Å from the aminoacylation active site in the canonical core of the enzyme, requiring translocation of mischarged amino acids for editing. An ensemble of crystal and cocrystal structures for LeuRS, IleRS, and ValRS suggests that the CP1 domain rotates via its flexible β-strand linkers relative to the main body along various steps in the enzyme's reaction pathway. Computational analysis suggested that the end of the N-terminal β-strand acted as a hinge. We hypothesized that a molecular hinge could specifically direct movement of the CP1 domain relative to the main body. We introduced a series of mutations into both β-strands in attempts to hinder movement and alter fidelity of LeuRS. Our results have identified specific residues within the β-strand tethers that selectively impact enzyme activity, supporting the idea that β-strand orientation is crucial for LeuRS canonical core and CP1 domain functions.