Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase.

Department of Biology, Faculty of Science, Niigata University, 8050 Ikarashi 2-no-cho, Nishi-ku, Niigata 950-2181 and Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
Nucleic Acids Research (Impact Factor: 8.81). 08/2012; DOI: 10.1093/nar/gks790
Source: PubMed

ABSTRACT Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-TΨC domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 Å resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies' results.

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    ABSTRACT: Peptidyl-tRNA hydrolase (Pth) catalyses the release of tRNA and peptide components from peptidyl-tRNA molecules. Pth from a Gram-positive bacterium Streptococcus pyogenes (SpPth) was cloned, expressed, purified and crystallised. Three-dimensional structure of SpPth was determined by X-ray crystallography at 2.19 Å resolution. Structure determination showed that the asymmetric unit of the unit cell contained two crystallographically independent molecules, designated A and B. The superimposition of C(α) traces of molecules A and B showed an r.m.s. shift of 0.4 Å, indicating that the structures of two crystallographically independent molecules were identical. The polypeptide chain of SpPth adopted an overall α/β conformation. The substrate-binding cleft in SpPth is formed with three loops: the gate loop, Ile91-Leu102; the base loop, Gly108-Gly115; and the lid loop, Gly136-Gly150. Unlike in the structures of Pth from Gram-negative bacteria, the entry to the cleft in the structure of SpPth appeared to be virtually closed. However, the conformations of the active site residues were found to be similar.
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