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.

  • Compound

    phosphotyrosine

  • Compound

    4-phosphomethyl-L-phenylalanine

  • Compound

    (S)-2-((S)-2,6-diaminohexanamido)-3-(4-(phosphonooxy)phenyl)propanoic acid

  • Compound

    (S)-2-((S)-2,6-diaminohexanamido)-3-(4-(phosphonomethyl)phenyl)propanoic acid

  • Compound

    4-(carboxymethyl)phenylalanine

  • Compound

    p-boronophenylalanine

  • Compound

    sulfotyrosine

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Protein Data Bank

Referenced accessions

References

  1. 1.

    , , , & The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

  2. 2.

    Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191–203 (2004).

  3. 3.

    & Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 897–930 (1985).

  4. 4.

    et al. Protein tyrosine phosphatases in the human genome. Cell 117, 699–711 (2004).

  5. 5.

    , & An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell 18, 925–933 (1979).

  6. 6.

    , , , & Regulation of the low molecular weight phosphotyrosine phosphatase by phosphorylation at tyrosines 131 and 132. J. Biol. Chem. 272, 5371–5374 (1997).

  7. 7.

    , & SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259, 1607–1611 (1993).

  8. 8.

    et al. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis. Nat. Chem. Biol. 8, 262–269 (2012).

  9. 9.

    & Synthetic approaches to protein phosphorylation. Curr. Opin. Chem. Biol. 28, 115–122 (2015).

  10. 10.

    et al. Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew. Chem. Int. Ed. Engl. 48, 8234–8239 (2009).

  11. 11.

    et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

  12. 12.

    , , & Structurally modified firefly luciferase. Effects of amino acid substitution at position 286. J. Am. Chem. Soc. 119, 10877–10887 (1997).

  13. 13.

    & Recombinant expression of selectively sulfated proteins in Escherichia coli. Nat. Biotechnol. 24, 1436–1440 (2006).

  14. 14.

    et al. Protein evolution with an expanded genetic code. Proc. Natl. Acad. Sci. USA 105, 17688–17693 (2008).

  15. 15.

    , , , & Mutagenesis and evolution of sulfated antibodies using an expanded genetic code. Biochemistry 48, 8891–8898 (2009).

  16. 16.

    , , & Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code. Nat. Protoc. 4, 1784–1789 (2009).

  17. 17.

    , , & Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin. J. Am. Chem. Soc. 129, 10648–10649 (2007).

  18. 18.

    , & A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli. ACS Chem. Biol. 2, 474–478 (2007).

  19. 19.

    et al. Using unnatural amino acid mutagenesis to probe the regulation of PRMT1. ACS Chem. Biol. 9, 649–655 (2014).

  20. 20.

    et al. Mimicking tyrosine phosphorylation in human cytochrome c by the evolved tRNA synthetase technique. Chemistry 21, 15004–15012 (2015).

  21. 21.

    , & A genetically encoded sulfotyrosine for VHR function research. Protein Cell 4, 731–734 (2013).

  22. 22.

    , & Chemical dissection of the effects of tyrosine phosphorylation of SHP-2. Biochemistry 42, 5461–5468 (2003).

  23. 23.

    , , & The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation. J. Biol. Chem. 278, 4668–4674 (2003).

  24. 24.

    , , & Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling. Mol. Cell 8, 759–769 (2001).

  25. 25.

    A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. USA 53, 745–750 (1965).

  26. 26.

    & Transport of impermeant substances in E. coli by way of oligopeptide permease. Nat. New Biol. 241, 161–163 (1973).

  27. 27.

    , , , & Illicit transport: the oligopeptide permease. Proc. Natl. Acad. Sci. USA 70, 456–458 (1973).

  28. 28.

    et al. Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem. Biol. 7, 1292–1302 (2012).

  29. 29.

    & The role of the terminal carboxyl group on peptide transport in Escherichia coli. J. Biol. Chem. 243, 335–340 (1968).

  30. 30.

    , , , & The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli. J. Bacteriol. 175, 1452–1456 (1993).

  31. 31.

    et al. A genetically encoded boronate-containing amino acid. Angew. Chem. Int. Ed. Engl. 47, 8220–8223 (2008).

  32. 32.

    , , & An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374 (2010).

  33. 33.

    & Parallel chemical protein synthesis on a surface enables the rapid analysis of the phosphoregulation of SH3 domains. Angew. Chem. Int. Ed. Engl. 55, 7252–7256 (2016).

  34. 34.

    , , , & A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52, 1828–1837 (2013).

  35. 35.

    , , , & Characterization of the interaction of natural proline-rich peptides with five different SH3 domains. Biochemistry 33, 10925–10933 (1994).

  36. 36.

    et al. An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity. Biochemistry 50, 1894–1900 (2011).

  37. 37.

    , , & Current chemical biology tools for studying protein phosphorylation and dephosphorylation. Chemistry 18, 28–39 (2012).

  38. 38.

    & Progress toward the evolution of an organism with an expanded genetic code. Proc. Natl. Acad. Sci. USA 96, 4780–4785 (1999).

  39. 39.

    et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science 333, 1151–1154 (2011).

  40. 40.

    , & Expanding the genetic code of Escherichia coli with phosphotyrosine. FEBS Lett. 590, 3040–3047 (2016).

  41. 41.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  42. 42.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  43. 43.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  44. 44.

    et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol. Crystallogr. 60, 2184–2195 (2004).

  45. 45.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

  46. 46.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

Download references

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.

Author information

Author notes

    • Rongsheng E Wang

    Present address: Department of Chemistry, Temple University, Philadelphia, Pennsylvania, USA.

Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.

    • Xiaozhou Luo
    • , Claudio Zambaldo
    • , Tao Liu
    • , Weimin Xuan
    • , Anzhi Yao
    • , Sean A Reed
    • , Mingchao Kang
    • , Chunhui Huang
    • , Peng-Yu Yang
    •  & Peter G Schultz
  2. Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Xiaozhou Luo
    • , Rongsheng E Wang
    • , Claudio Zambaldo
    • , Tao Liu
    • , Weimin Xuan
    • , Anzhi Yao
    • , Sean A Reed
    • , Mingchao Kang
    • , Chunhui Huang
    • , Peng-Yu Yang
    • , Ian A Wilson
    •  & Peter G Schultz
  3. California Institute for Biomedical Research (Calibr), La Jolla, California, USA.

    • Guangsen Fu
    • , Renhe Liu
    • , Xiaoxuan Lyu
    • , Jintang Du
    • , Yuhan Zhang
    • , Hui Guo
    • , Peter G Schultz
    •  & Feng Wang
  4. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Xueyong Zhu
    •  & Ian A Wilson

Authors

  1. Search for Xiaozhou Luo in:

  2. Search for Guangsen Fu in:

  3. Search for Rongsheng E Wang in:

  4. Search for Xueyong Zhu in:

  5. Search for Claudio Zambaldo in:

  6. Search for Renhe Liu in:

  7. Search for Tao Liu in:

  8. Search for Xiaoxuan Lyu in:

  9. Search for Jintang Du in:

  10. Search for Weimin Xuan in:

  11. Search for Anzhi Yao in:

  12. Search for Sean A Reed in:

  13. Search for Mingchao Kang in:

  14. Search for Yuhan Zhang in:

  15. Search for Hui Guo in:

  16. Search for Chunhui Huang in:

  17. Search for Peng-Yu Yang in:

  18. Search for Ian A Wilson in:

  19. Search for Peter G Schultz in:

  20. Search for Feng Wang in:

Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Peter G Schultz or Feng Wang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–7

  2. 2.

    Supplementary Note

    Supplementary Procedures

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.2405

Further reading

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing