Abstract
Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase–tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.
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Acknowledgements
The authors acknowledge K. Williams for the assistance in manuscript preparation. X-ray diffraction data were collected at the Advanced Photon Source (APS) beamline 23ID-B. Use of the Advanced Photon Source for data collection was supported by the DOE, Basic Energy Sciences, Office of Science, under contract no. DE-AC02- 06CH11357. GM/CA CAT has been funded in whole or in part with federal funds from NCI (grant Y1-CO-1020) and NIGMS (grant Y1-GM-1104). The NIH and DOE funders at the beamlines had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by NIH Grant 5R01 GM062159-14 (to P.G.S). This is manuscript 29424 of The Scripps Research Institute.
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X. Luo, P.G.S., and F.W. designed the research. X. Luo, G.F., and R.E.W. performed protein expression, purification, and crystallization. X. Luo, R.E.W., C.Z., R.L., W.X., C.H. and P.-Y.Y. performed chemical synthesis. X. Luo, T.L., J.D., M.K., and Y.Z. performed the cloning and screening of synthetases, expression of target proteins. X.Z. performed X-ray diffraction experiments.; X. Luo, G.F., X.Z., X. Lyu., I.A.W. and F.W. performed crystallographic analysis and data deposition. X. Luo, H.G., and A.Y. performed fluorescence polarization assay. X. Luo, T.L., W.X., P.G.S. and F.W. analyzed the data; and X. Luo, S.A.R., P.G.S. and F.W. wrote the paper.
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Luo, X., Fu, G., Wang, R. et al. Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria. Nat Chem Biol 13, 845–849 (2017). https://doi.org/10.1038/nchembio.2405
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DOI: https://doi.org/10.1038/nchembio.2405
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