Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from<i>Escherichia coli</i>in complex with the acceptor-TΨC domain of tRNA

Ito, Kosuke; Hao, Qian; Shimizu, Yoshihiro; Murakami, Ryo; Miura, Koryo; Ueda, Takuya; Uchiumi, Toshio

doi:10.1107/s1744309111038383

Cited by 11 publications

(9 citation statements)

References 33 publications

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“…Sample production, purification and crystallization of E. coli Pth in complex with the CCA - acceptor-TΨC domain of E. coli tRNA Ala (G1 to A7 and A49 to A76 of E. coli ) were performed as previously described ( 29 ). Briefly, E. coli Pth was overexpressed in E. coli strain BL21(DE3) with an N-terminal His-tag.…”

Section: Methodsmentioning

confidence: 99%

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

Ito

Murakami

Mochizuki

et al. 2012

Nucleic Acids Research

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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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Section: Methodsmentioning

confidence: 99%

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

Ito

Murakami

Mochizuki

et al. 2012

Nucleic Acids Research

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“…The Tt Pth used in this experiment was prepared as described above. Ec Pth was prepared as described previously . Melt curves were obtained using the StepOne Real‐Time PCR system (ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

High‐resolution crystal structure of peptidyl‐tRNA hydrolase from Thermus thermophilus

et al. 2018

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Peptidyl‐tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl‐tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N‐ and C‐terminal, loop‐helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

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“…Helix 4 occludes three residues, Asn10, His20 and Asp93 (as numbered in Escherichia coli Pth1), identified by site-directed mutagenesis to be crucially involved in enzyme activity (Goodall et al, 2004). The structure of E. coli Pth1 in the complex of the T C loop of tRNA has recently been reported and has provided invaluable structural insight into the nucleotide binding (Ito et al, 2011). An overall shape of the complex furthers understanding (Hames et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase fromSalmonella typhimurium

et al. 2014

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PDB reference: peptidyl-tRNA hydrolase, 4p7bPeptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacterium Salmonella typhimurium has been cloned, expressed in Escherichia coli and crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 M citric acid, 0.05 M bistris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P2 1 2 1 2 1 with unit-cell parameters a = 62.1, b = 64.9, c = 110.5 Å , = = = 90. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å 3 Da À1 . The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.

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Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase fromEscherichia coliin complex with the acceptor-TΨC domain of tRNA

Cited by 11 publications

References 33 publications

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

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

High‐resolution crystal structure of peptidyl‐tRNA hydrolase from Thermus thermophilus

Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase fromSalmonella typhimurium

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