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 are multidomain proteins that catalyze the covalent attachment of amino acids to their cognate transfer RNA. Various domains of an aminoacyl-tRNA synthetase perform their specific functions in a highly coordinated manner to maintain high accuracy in protein synthesis in cells. The coordination of their function, therefore, requires communication between domains. In this study we explored the relevance of enzyme motion in domain-domain communications. Specifically, we attempted to probe whether the communication between distantly located domains of a multidomain protein is accomplished through a coordinated movement of structural elements. We investigated the collective motion in Thermus thermophilus leucyl-tRNA synthetase by studying the low frequency normal modes. We identified the mode that best described the experimentally observed conformational changes of T. thermophilus leucyl-tRNA synthetase upon substrate binding and analyzed the correlated and anticorrelated motions between different domains. Furthermore, we used statistical coupling analysis to explore if the amino acid pairs and/or clusters whose motions are thermally coupled have also coevolved. Our study demonstrates that a small number of residues belong to the category whose coupled thermal motions correspond to evolutionary coupling as well. These residue clusters constitute a distinguished set of interacting networks that are sparsely distributed in the domain interface. Residues of these networking clusters are within van der Waals contact, and we suggest that they are critical in the propagation of long range mechanochemical motions in T. thermophilus leucyl-tRNA synthetase.
    Preview · Article · Mar 2009 · Journal of Biological Chemistry
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    ABSTRACT: Quality control mechanisms during protein synthesis are essential to fidelity and cell survival. Leucyl-tRNA synthetase (LeuRS) misactivates non-leucine amino acids including isoleucine, methionine, and norvaline. To prevent translational errors, mischarged tRNA products are translocated 30A from the canonical aminoacylation core to a hydrolytic editing-active site within a completely separate domain. Because it is transient, the tRNA translocation mechanism has been difficult to isolate. We have identified a "translocation peptide" within Escherichia coli LeuRS. Mutations in the translocation peptide cause tRNA to selectively bypass the editing-active site, resulting in mischarging that is lethal to the cell. This bypass mechanism also rescues aminoacylation of an editing site mutation that hydrolyzes correctly charged Leu-tRNA(Leu). Thus, these LeuRS mutants charge tRNA(Leu) but fail to translocate these products to the hydrolytic site, where they are cleared to guard against genetic code ambiguities.
    No preview · Article · Apr 2009 · Journal of Biological Chemistry
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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.
    Preview · Article · Sep 2009 · FEBS letters
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