Idiosyncrasy and identity in the prokaryotic phe‐system: Crystal structure of <i>E. coli</i> phenylalanyl‐tRNA synthetase complexed with phenylalanine and AMP

Mermershtain, Inbal; Finarov, Igal; Klipcan, Liron; Kessler, Naama; Rozenberg, Haim; Safro, Mark

doi:10.1002/pro.549

Cited by 46 publications

(59 citation statements)

References 35 publications

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“…2A and SI Appendix, Fig. S1) in the alpha subunit has been proposed to play a key role in determining substrate specificity in both prokaryotic and eukaryotic PheRSs (9,10). Therefore, we hypothesized that mutating this residue to smaller residues should enable CePheRS to activate and charge the larger azide-bearing Phe analog Azf to CetRNA Phe .…”

Section: Resultsmentioning

confidence: 99%

Cell-specific proteomic analysis in Caenorhabditis elegans

Yuet¹,

Doma

Ngo³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.click chemistry | protein engineering | proteomics | cell-specific protein expression | nematode pharyngeal muscle I n a complex eukaryote like Caenorhabditis elegans, cell heterogeneity restricts the usefulness of large-scale, mass spectrometry-based proteomic analysis. Enriching for specific cells is challenging, and researchers cannot systematically identify low-abundance proteins expressed in specific cells from wholeorganism lysates. Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1, 2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing L-methionine (Met) analog L-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3, 4). Because Anl is a poor substrate for any of the natural aminoacyl-tRNA synthetases, it is excluded from proteins made in wild-type cells but is incorporated readily into proteins made in cells that express an appropriately engineered MetRS. Controlling expression of mutant MetRSs by expression only in specific cells restricts Anl labeling to proteins produced in those cells. Tagged proteins can be distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to alkynyl or cyclooctynyl probes that permit facile detection, isolation, and visualization of labeled proteins. This strategy has been used to selectively enrich microbial proteins from mixtures of bacterial and mammalian cells. For example, Ngo et al. (5) found that proteins made in an E. coli strain outf...

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Section: Resultsmentioning

confidence: 99%

Cell-specific proteomic analysis in Caenorhabditis elegans

Yuet¹,

Doma

Ngo³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Crystal Structure of P. aeruginosa PheRS-A variety of PheRS crystal structures from E. coli and Thermus thermophilus have been previously described, including the apo enzyme, complexes with either tRNA Phe or the substrates phenylalanine and AMP, and with the phenylalanyl-adenylate intermediate (12,(42)(43)(44). Moreover, two PheRS structures from the Gram-positive pathogen S. haemolyticus with a phenyl-thiazolylurea-sulfonamide inhibitor (compound 1a) have been determined (27).…”

Section: Phenyl-thiazolylurea-sulfonamides Act Via Phers In Gram-mentioning

confidence: 99%

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Abibi

Ferguson

Fleming

et al. 2014

Journal of Biological Chemistry

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Background: Phenylalanyl-tRNA synthetase inhibitors have been shown to be efficacious in animal models of infection. Results: Inhibitors occupy a newly identified hydrophobic auxiliary binding pocket. Conclusion: Compound binding in this pocket leads to high screening hit rates, resistance frequencies, and elevated plasma protein binding. Significance: New inhibitors may be identified by avoiding the auxiliary pocket.

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“…In view of close structural similarity between EcPheRS and TtPheRS (11,20), and the better resolution limit observed for crystals of the thermophilic homolog, we determined the crystal structure of TtPheRS in complex with puromycin and phenylalanine at 2.6 Å resolution (Table S1). Crystals of TtPheRS were grown as described previously (21).…”

Section: Resultsmentioning

confidence: 99%

“…The E. coli PheRS was cloned, expressed, and purified as described previously (20). TtPheRS was purified and crystallized as described (25).…”

Section: Methodsmentioning

confidence: 99%

Universal pathway for posttransfer editing reactions: Insights from the crystal structure of Tt PheRS with puromycin

Tworowski

Klipcan

Peretz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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At the amino acid binding and recognition step, phenylalanyltRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA Phe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis-and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA Phe where deacylation occurs from either the 2′-or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.biosynthesis | aminoacyl-tRNA synthetases | tRNA | puromycin | editing

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Idiosyncrasy and identity in the prokaryotic phe‐system: Crystal structure of E. coli phenylalanyl‐tRNA synthetase complexed with phenylalanine and AMP

Cited by 46 publications

References 35 publications

Cell-specific proteomic analysis in Caenorhabditis elegans

Cell-specific proteomic analysis in Caenorhabditis elegans

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Universal pathway for posttransfer editing reactions: Insights from the crystal structure of Tt PheRS with puromycin

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