Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Palladium-triggered deprotection chemistry for protein activation in living cells

Nature Chemistry volume 6, pages 352–361 (2014)Cite this article

Subjects

Abstract

Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A chemical decaging strategy for protein activation in living cells.
Figure 2: Catalyst screening for palladium-mediated deprotection reactions.
Figure 3: Identification of biocompatible and efficient palladium reagents for propargyl carbamate cleavage on intact proteins.
Figure 4: Palladium-mediated propargyl carbamate cleavage within living cells.
Figure 5: Modulating the activity of a bacterial phosphothreonine lyase OspF via palladium-mediated decaging of lysine in vitro and in vivo.
Figure 6: Chemically rescued OspF as a tool to study the intracellular localization of its cognitive substrate Erk.

References

  1. Zorn, J. A. & Wells, J. A. Turning enzymes ON with small molecules. Nature Chem. Biol. 6, 179–188 (2010).

    Article  CAS  Google Scholar 

  2. Bishop, A. & Chen, V. Brought to life: targeted activation of enzyme function with small molecules. J. Chem. Biol., 2, 1–9 (2009).

    Article  Google Scholar 

  3. Riggsbee, C. W. & Deiters, A. Recent advances in the photochemical control of protein function. Trends Biotechnol. 28, 468–475 (2010).

    Article  CAS  Google Scholar 

  4. Banaszynski, L. A., Chen, L-C., Maynard-Smith, L. A., Ooi, A. G. L. & Wandless, T. J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    Article  CAS  Google Scholar 

  5. Ghosh M. et al. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304, 743–746 (2004).

    Article  CAS  Google Scholar 

  6. Wolan, D. W., Zorn, J. A., Gray, D. C. & Wells, J. A. Small-molecule activators of a proenzyme. Science 326, 853–858 (2009).

    Article  CAS  Google Scholar 

  7. Erster O., Eisenstein, M. & Liscovitch, M. Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles. Nature Methods 4, 393–395 (2007).

    Article  CAS  Google Scholar 

  8. Qiao, Y., Molina, H., Pandey, A., Zhang. J. & Cole, P. A. Chemical rescue of a mutant enzyme in living cells. Science 311, 1293–1297 (2006).

    Article  CAS  Google Scholar 

  9. Pratt, M. R, Schwartz, E. C. & Muir, T. W. Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt. Proc. Natl Acad. Sci. USA 104, 11209–11214 (2007).

    Article  CAS  Google Scholar 

  10. Li, H., Hah, J-M. & Lawrence, D. S. Light-mediated liberation of enzymatic activity: ‘small molecule’ caged protein equivalents. J. Am. Chem. Soc. 130, 10474–10475 (2008).

    Article  CAS  Google Scholar 

  11. Zou, K., Cheley, S., Givens, R. S. & Bayley, H. Catalytic subunit of protein kinase a caged at the activating phosphothreonine. J. Am. Chem. Soc. 124, 8220–8229 (2002).

    Article  CAS  Google Scholar 

  12. Chen, P. R. et al. A facile system for encoding unnatural amino acids in mammalian cells. Angew. Chem. Int. Ed. 48, 4052–4055 (2009).

    Article  CAS  Google Scholar 

  13. Davis, L. & Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nature Rev. Mol. Cell Biol. 13, 168–182 (2012).

    Article  CAS  Google Scholar 

  14. Arbely, E., Torres-Kolbus, J., Deiters, A. & Chin, J. W. Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine. J. Am. Chem. Soc. 134, 11912–11915 (2012).

    Article  CAS  Google Scholar 

  15. Oksvold, M. P., Huitfeldt, H. S., Østvold, A. C. & Skarpen, E. UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor. J. Cell Sci. 115, 793–803 (2002).

    CAS  PubMed  Google Scholar 

  16. Walsh, C. T. Posttranslational Modification of Proteins: Expanding Nature's Inventory 1st edn (Roberts and Company, 2005).

    Google Scholar 

  17. Carrera, A. C., Alexandrov, K. & Roberts, T. M. The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP. Proc. Natl Acad. Sci. USA 90, 442–446 (1993).

    Article  CAS  Google Scholar 

  18. Streu, C. & Meggers, E. Ruthenium-induced allylcarbamate cleavage in living cells. Angew. Chem. Int. Ed. 45, 5645–5648 (2006).

    Article  CAS  Google Scholar 

  19. Garner, A. L., Song, F. & Koide, K. Enhancement of a catalysis-based fluorometric detection method for palladium through rational fine-tuning of the palladium species. J. Am. Chem. Soc. 131, 5163–5171 (2009).

    Article  CAS  Google Scholar 

  20. Isidro-Llobet, A., Álvarez, M. & Albericio, F. Amino acid-protecting groups. Chem. Rev. 109, 2455–2504 (2009).

    Article  CAS  Google Scholar 

  21. Ai, H-W., Lee, J. W. & Schultz, P. G. A method to site-specifically introduce methyllysine into proteins in E. coli. Chem. Commun. 46, 5506–5508 (2010).

    Article  CAS  Google Scholar 

  22. Santra, M., Ko, S.-K., Shin, I & Ahn, K. H. Fluorescent detection of palladium species with an O-propargylated fluorescein. Chem. Commun. 46, 3964–3966 (2010).

    Article  CAS  Google Scholar 

  23. Yusop, R. M., Unciti-Broceta, A., Johansson, E. M. V., Sánchez-Martín, R. M. & Bradley, M. Palladium-mediated intracellular chemistry. Nature Chem. 3, 239–243 (2011).

    Article  CAS  Google Scholar 

  24. Sasmal, P. K., Streu, C. N. & Meggers, E. Metal complex catalysis in living biological systems. Chem. Commun. 49, 1581–1587 (2013).

    Article  CAS  Google Scholar 

  25. Li. J. et al. Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens. J. Am. Chem. Soc. 135, 7330–7338 (2013).

    Article  CAS  Google Scholar 

  26. Chalker, J. M., Wood, C. S. C. & Davis, B. G. A convenient catalyst for aqueous and protein Suzuki–Miyaura cross-coupling. J. Am. Chem. Soc. 131, 16346–16347 (2009).

    Article  CAS  Google Scholar 

  27. Fors, B. P., Krattiger, P., Strieter, E. & Buchwald, S. L. Water-mediated catalyst preactivation: an efficient protocol for C–N cross-coupling reactions. Org. Lett. 10, 3505–3508 (2008).

    Article  CAS  Google Scholar 

  28. Ito, T., Takahashi, Y. & Ishii Y. Convenient preparation of novel palladium-π-olefin complexes from bis(dibenzylideneacetone)palladium(0). Chem. Commun. 629a–629a (1972).

  29. Wei, C. S. et al. The impact of palladium(II) reduction pathways on the structure and activity of palladium(0) catalysts. Angew. Chem. Int. Ed. 52, 5822–5826 (2013).

    Article  CAS  Google Scholar 

  30. Li, N., Lim, R. K. V., Edwardraja, S. & Lin, Q. Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells. J. Am. Chem. Soc. 133, 15316–15319 (2011).

    Article  CAS  Google Scholar 

  31. Rambabu, D., Bhavani, S., Swamy, N. K., Basaveswara Rao, M. V. & Pal, M. Pd/C-mediated depropargylation of propargyl ethers/amines in water. Tetrahedron Lett. 54, 1169–1173 (2013).

    Article  CAS  Google Scholar 

  32. Hao, B. et al. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466 (2002).

    Article  CAS  Google Scholar 

  33. Mukai T et al. Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

    Article  CAS  Google Scholar 

  34. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  Google Scholar 

  35. Bianco, A., Townsley, F. M., Greiss, S., Lang, K. & Chin, J. W. Expanding the genetic code of Drosophila melanogaster. Nature Chem. Biol. 8, 748–750 (2012).

    Article  CAS  Google Scholar 

  36. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNACUA pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009).

    Article  CAS  Google Scholar 

  37. Kennedy, D. C. et al. Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions. J. Am. Chem. Soc. 133, 17993–18001 (2011).

    Article  CAS  Google Scholar 

  38. Li, H. et al. The phosphothreonine lyase activity of a bacterial Type III effector family. Science 315, 1000–1003 (2007).

    Article  CAS  Google Scholar 

  39. Arbibe, L. et al. An injected bacterial effector targets chromatin access for transcription factor NF-κB to alter transcription of host genes involved in immune responses. Nature Immunol. 8, 47–56 (2007).

    Article  CAS  Google Scholar 

  40. Zhu, Y. et al. Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase. Mol. Cell 28, 899–913 (2007).

    Article  CAS  Google Scholar 

  41. Lidke, D. S. et al. ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation. J. Biol. Chem. 285, 3092–3102 (2010).

    Article  CAS  Google Scholar 

  42. Volmat, V., Camps, M., Arkinstall, S., Pouysségur, J. & Lenormand, P. The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases. J. Cell Sci. 114, 3433–3443 (2001).

    CAS  PubMed  Google Scholar 

  43. Wei, P. et al. Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells. Nature 488, 384–388 (2012).

    Article  CAS  Google Scholar 

  44. Chuikov, S. et al. Regulation of p53 activity through lysine methylation. Nature 432, 353–360 (2004).

    Article  CAS  Google Scholar 

  45. Hendrickx, A. P. A., Budzik, J. M., Oh, S-Y. & Schneewind, O. Architects at the bacterial surface – sortases and the assembly of pili with isopeptide bonds. Nature Rev. Micro. 9, 166–176 (2011).

    Article  CAS  Google Scholar 

  46. Li, J. & Chen, P. R. Moving Pd-mediated protein cross coupling to living systems. Chembiochem 13, 1728–1731 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by research grants from National Natural Science Foundation of China (21225206 and 91313301) and the National Key Basic Research Foundation of China (2010CB912302). We thank F. Shao and O. Schneewind for the donation of plasmids.

Author information

Authors and Affiliations

  1. Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China

    Jie Li, Jingyi Zhao, Siqi Zheng, Shixian Lin, Long Chen, Maiyun Yang, Shang Jia & Peng R. Chen

  2. Peking-Tsinghua Center for Life Sciences, Beijing 100871, China

    Juntao Yu, Jie Wang & Peng R. Chen

  3. College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China

    Xiaoyu Zhang

Authors
  1. Jie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juntao Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingyi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jie Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Siqi Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shixian Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Long Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Maiyun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shang Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiaoyu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Peng R. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.R.C. conceived and designed the experiments. J.L., J.Y., J.Z., J.W., S.Z., S.L., L.C., M.Y., S.J. and X.Z. performed the experiments. P.R.C., J.L., J.Y., J.Z. and J.W. analysed the data. P.R.C, J.L. and J.Y. prepared the figures and co-wrote the paper, with input from all the authors.

Corresponding author

Correspondence to Peng R. Chen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 8222 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, J., Yu, J., Zhao, J. et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nature Chem 6, 352–361 (2014). https://doi.org/10.1038/nchem.1887

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1887

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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