Functional Segregation of a Predicted “Hinge” Site within the β-Strand Linkers of Escherichia coli Leucyl-tRNA Synthetase †

Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801-3732, USA.
Biochemistry (Impact Factor: 3.02). 05/2008; 47(16):4808-16. DOI: 10.1021/bi702494q
Source: PubMed


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 beta-strand tethers. The CP1 hydrolytic editing active site is located approximately 30 A 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 beta-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 beta-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 beta-strands in attempts to hinder movement and alter fidelity of LeuRS. Our results have identified specific residues within the beta-strand tethers that selectively impact enzyme activity, supporting the idea that beta-strand orientation is crucial for LeuRS canonical core and CP1 domain functions.

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    ABSTRACT: Aminoacyl-tRNA synthetases often rely on a proofreading mechanism to clear mischarging errors before they can be incorporated into newly synthesized proteins. Leucyl-tRNA synthetase (LeuRS) houses a hydrolytic editing pocket in a domain that is distinct from its aminoacylation domain. Mischarged amino acids are transiently translocated approximately 30A between active sites for editing by an unknown tRNA-dependent mechanism. A glycine within a flexible beta-strand that links the aminoacylation and editing domains of LeuRS was determined to be important to tRNA translocation. The translocation-defective mutation also demonstrated that the editing site screens both correctly and incorrectly charged tRNAs prior to product release.
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