Structure of Human Cytosolic Phenylalanyl-tRNA Synthetase: Evidence for Kingdom-Specific Design of the Active Sites and tRNA Binding Patterns

Finarov, Igal; Moor, Nina; Kessler, Naama; Klipcan, Liron; Safro, Mark

doi:10.1016/j.str.2010.01.002

Cited by 59 publications

(77 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

Cell-specific proteomic analysis in Caenorhabditis elegans

Yuet¹,

Doma

Ngo³

et al. 2015

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

View full text Add to dashboard Cite

show abstract

“…26 Apart from difference in length, there is also marked divergence in domain organization between the two kingdoms. 27,28 Conformational switch: "non-active" to "active" state α or β subunit on its own is catalytically inactive. 29,30 Affinity labeling to localize the substrate binding site in Escherichia coli PheRS (ecPheRS) along with crystallographic data of Thermus thermophilus PheRS (ttPheRS) complexed with phenylalanine (Phe) and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analog of phenylalanyl-adenylate (Phe-AMP), corroborate that the α subunit of PheRS on its own is unable to carry out the first step of the aminoacylation reaction.…”

Section: Structural Diversity Of Phersmentioning

confidence: 99%

“…Similarly, the helical loop in HctPheRS containing a highly conserved sequence [xProxxHisProAlaArgAsp(Met/x)(Trp/Gln/His) AspThrPhe] is important for the proper positioning of CCA end of tRNA Phe . 27 The tRNA…”

Section: Structural Diversity Of Phersmentioning

confidence: 99%

“…Two different α subunits of PheRS are involved in the acceptor end and the anticodon stem binding of a tRNA Phe . 27,32 Crystal structures clearly depict that the heterotetrameric PheRS is able to bind two molecules of tRNA Phe simultaneously. 36 Organelle-specific PheRS: a chimera…”

Section: T T T T T T T Tyr Yr Tyr Tyr Yr T T T T T Tyr T T T T T T Tymentioning

confidence: 99%

“…35 In HctPheRS, βGlu254, and βAsn238 corresponding to βGlu334 and βHis261 in ttPheRS respectively, play a crucial role in recognition and editing of the Tyr moiety. 27 The corresponding residues, which are crucial for editing in E. coli are βGlu334 and βHis265 62 and in yeast are Aspβ243 and Hisβ158. 65 The B2 domain has also been shown to play a key role in post-transfer editing.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Phenylalanyl-tRNA synthetase

Chakraborty¹,

Banerjee²

2016

RRBC

View full text Add to dashboard Cite

Phenylalalnyl-tRNA synthetase (PheRS), a member of class II aminoacyl-tRNA synthetases, catalyzes the synthesis of phenylalanyl-tRNA Phe (Phe-tRNA Phe ). Hence, like other aminoacyl-tRNA synthetases, PheRS also plays a crucial role in the cellular translation process. Structural characterization demonstrates remarkable architectural diversity ranging from monomer to hetero-oligomer. Heterotetrameric PheRS contains an editing domain to proofread misincorporation of non-cognate amino acids. However, editing activity is absent in monomeric PheRS. PheRS has also shown some noncanonical functions, such as DNA binding properties, suggesting its involvement in complex regulatory pathways. Engineered mutants with relaxed substrate specificities of PheRS can be a promising tool for chemical and synthetic biology. Because of substantial structural variations among species due to evolutionary divergence, PheRS may be validated as a novel drug target. Human PheRS gene mutations have recently been implicated in several neurological disorders prompting structure-function studies to elucidate the molecular role of PheRS in such pathologies. In this review on PheRS, we will briefly cover all of the aforementioned aspects and our current understanding about the enzyme.

show abstract

Molecular polygamy: The promiscuity of l‐phenylalanyl‐tRNA‐synthetase triggers misincorporation ofmeta‐ andortho‐tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells

Popp¹,

Larraillet

Kettenberger

et al. 2015

Biotech & Bioengineering

View full text Add to dashboard Cite

In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention.

show abstract

Structure of Human Cytosolic Phenylalanyl-tRNA Synthetase: Evidence for Kingdom-Specific Design of the Active Sites and tRNA Binding Patterns

Cited by 59 publications

References 46 publications

Cell-specific proteomic analysis in Caenorhabditis elegans

Cell-specific proteomic analysis in Caenorhabditis elegans

Phenylalanyl-tRNA synthetase

Molecular polygamy: The promiscuity of l‐phenylalanyl‐tRNA‐synthetase triggers misincorporation ofmeta‐ andortho‐tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells

Contact Info

Product

Resources

About