NMR-Based Substrate Analog Docking to Escherichia coli Peptidyl-tRNA Hydrolase

Giorgi, Laurent; Plateau, Pierre; O’Mahony, Gavin; Aubard, Caroline; Fromant, Michel; Thureau, Aurélien; Grøtli, Morten; Blanquet, Sylvain; Bontems, François

doi:10.1016/j.jmb.2011.06.025

Cited by 21 publications

(28 citation statements)

References 36 publications

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“…This result suggested that a functional interaction with Pth could still be achieved, although the TC arm has been removed. In agreement with this view, we reported elsewhere Ϫ5 M Ϫ1 ⅐s Ϫ1 ) (24). However, we conclude here that the introduction of the TC arm in a putative substrate significantly improves the efficiency of catalysis.…”

Section: Mini-and Microhelixsupporting

confidence: 92%

“…Transformants were grown in 1 liter of 2ϫ TY-rich medium. Induction of the expression of the Pth variants and protein purification by affinity chromatography were performed as described previously for the production of His-tagged Pth H20A (24).…”

Section: Production and Purification Of Wild-type And Mutant Pths-mentioning

confidence: 99%

“…15 N-Labeled His-tagged H20A and H20A R133A Pth variants were produced in K37⌬recADE3 cells and purified by affinity chromatography as described previously in the case of the 15 N-labeled His-tagged H20A variant (24). Homogeneity of the samples was estimated to be higher than 95% by SDS-PAGE analysis.…”

Section: Production and Purification Of Wild-type And Mutant Pths-mentioning

confidence: 99%

“…Choice of Conditions of the NMR Study-Recently, we used NMR to gain some details of the binding of 3Ј-(L-[N,N-diacetyllysinyl])amino-3Ј-deoxyadenosine to E. coli Pth (24). To guard this ligand against hydrolysis under the NMR conditions, we used an inactive His-tagged H20A enzyme variant.…”

Section: Mini-and Microhelixmentioning

confidence: 99%

“…To guard this ligand against hydrolysis under the NMR conditions, we used an inactive His-tagged H20A enzyme variant. Here, to study the interaction between Pth and RNAs, we used the same His-tagged H20A variant because assignment of most of the proton resonances of this protein was available (in 50 mM phosphate buffer (pH 6.0) containing 200 mM NaCl, BMRB accession number 17404) (24). The His-20 residue plays an essential role in the hydrolysis reaction (8,29).…”

Section: Mini-and Microhelixmentioning

confidence: 99%

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RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

Giorgi

Bontems

Fromant

et al. 2011

Journal of Biological Chemistry

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Background: Bacterial peptidyl-tRNA hydrolase is essential in recycling of ribosome-dissociated peptidyl-tRNAs. Results: Comparing minimalist substrates and using NMR mapping, the RNA-binding site of the hydrolase is characterized. Conclusion: Interaction between the hydrolase and tRNA involves features common to all elongator tRNAs. Significance: Knowledge of a bacterial peptidyl-tRNA hydrolase⅐substrate complex may drive the search for enzyme inhibitors.

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Section: Mini-and Microhelixsupporting

confidence: 92%

Section: Production and Purification Of Wild-type And Mutant Pths-mentioning

confidence: 99%

Section: Production and Purification Of Wild-type And Mutant Pths-mentioning

confidence: 99%

Section: Mini-and Microhelixmentioning

confidence: 99%

Section: Mini-and Microhelixmentioning

confidence: 99%

See 3 more Smart Citations

RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

Giorgi

Bontems

Fromant

et al. 2011

Journal of Biological Chemistry

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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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Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

et al. 2016

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Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

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NMR-Based Substrate Analog Docking to Escherichia coli Peptidyl-tRNA Hydrolase

Cited by 21 publications

References 36 publications

RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

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

Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

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