Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase


Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.

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Fig. 1: General scheme showing sortase-mediated ubiquitylation (sortylation).
Fig. 2: Site-specific incorporation of AzGGK into proteins in E. coli.
Fig. 3: Srt5M-mediated ubiquitylation of GGK-bearing proteins.
Fig. 4: Srt2A-mediated ubiquitylation of GGK-bearing proteins.
Fig. 5: Site-specific SUMOylation of GGK-bearing proteins.
Fig. 6: Incorporation of AzGGK into proteins in mammalian cells and sortase-mediated ubiquitylation and SUMOylation of proteins in living HEK293T cells.

Data availability

The authors declare that the data supporting the findings of this study are available in the paper and its supplementary information files. Raw data and other materials are available upon reasonable request.


  1. 1.

    Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    CAS  Article  Google Scholar 

  2. 2.

    Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Kulathu, Y. & Komander, D. Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    CAS  Article  Google Scholar 

  4. 4.

    Komander, D., Clague, M. J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

    CAS  Article  Google Scholar 

  5. 5.

    van der Veen, A. G. & Ploegh, H. L. Ubiquitin-like proteins. Annu. Rev. Biochem. 81, 323–357 (2012).

    Article  Google Scholar 

  6. 6.

    Mali, S. M., Singh, S. K., Eid, E. & Brik, A. Ubiquitin signaling: chemistry comes to the rescue. J. Am. Chem. Soc. 139, 4971–4986 (2017).

    CAS  Article  Google Scholar 

  7. 7.

    Trang, V. H. et al. Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers. Angew. Chem. Int. Ed. Engl. 51, 13085–13088 (2012).

    CAS  Article  Google Scholar 

  8. 8.

    Chen, J., Ai, Y., Wang, J., Haracska, L. & Zhuang, Z. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat. Chem. Biol. 6, 270–272 (2010).

    CAS  Article  Google Scholar 

  9. 9.

    Weikart, N. D. & Mootz, H. D. Generation of site-specific and enzymatically stable conjugates of recombinant proteins with ubiquitin-like modifiers by the Cu(i)-catalyzed azide-alkyne cycloaddition. Chembiochem 11, 774–777 (2010).

    CAS  Article  Google Scholar 

  10. 10.

    Eger, S., Scheffner, M., Marx, A. & Rubini, M. Synthesis of defined ubiquitin dimers. J. Am. Chem. Soc. 132, 16337–16339 (2010).

    CAS  Article  Google Scholar 

  11. 11.

    Stanley, M. & Virdee, S. Genetically directed production of recombinant, isosteric and nonhydrolysable ubiquitin conjugates. Chembiochem 17, 1472–1480 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Virdee, S., Ye, Y., Nguyen, D. P., Komander, D. & Chin, J. W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

    CAS  Article  Google Scholar 

  13. 13.

    Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem. Int. Ed. Engl. 48, 9184–9187 (2009).

    CAS  Article  Google Scholar 

  14. 14.

    Virdee, S. et al. Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. 133, 10708–10711 (2011).

    CAS  Article  Google Scholar 

  15. 15.

    Stanley, M. & Virdee, S. Chemical ubiquitination for decrypting a cellular code. Biochem. J. 473, 1297–1314 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Spasser, L. & Brik, A. Chemistry and biology of the ubiquitin signal. Angew. Chem. Int. Ed. Engl. 51, 6840–6862 (2012).

    CAS  Article  Google Scholar 

  17. 17.

    Pham, G. H. & Strieter, E. R. Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin-protein conjugates. Curr. Opin. Chem. Biol. 28, 57–65 (2015).

    CAS  Article  Google Scholar 

  18. 18.

    Mazmanian, S. K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 (1999).

    CAS  Article  Google Scholar 

  19. 19.

    Popp, M. W. & Ploegh, H. L. Making and breaking peptide bonds: protein engineering using sortase. Angew. Chem. Int. Ed. Engl. 50, 5024–5032 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Chen, I., Dorr, B. M. & Liu, D. R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl Acad. Sci. USA 108, 11399–11404 (2011).

    CAS  Article  Google Scholar 

  21. 21.

    Luo, J., Liu, Q., Morihiro, K. & Deiters, A. Small-molecule control of protein function through Staudinger reduction. Nat. Chem. 8, 1027–1034 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    CAS  Article  Google Scholar 

  23. 23.

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

    CAS  Article  Google Scholar 

  24. 24.

    Hancock, S. M., Uprety, R., Deiters, A. & Chin, J. W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 4, 298–304 (2012).

    CAS  Article  Google Scholar 

  26. 26.

    Li, F. et al. Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana. Angew. Chem. Int. Ed. Engl. 52, 9700–9704 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Greiss, S. & Chin, J. W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 14196–14199 (2011).

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 29.

    Han, S. et al. Expanding the genetic code of Mus musculus. Nat. Commun. 8, 14568 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).

    CAS  Article  Google Scholar 

  31. 31.

    Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N(ε)-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008).

    CAS  Article  Google Scholar 

  32. 32.

    Dorr, B. M., Ham, H. O., An, C., Chaikof, E. L. & Liu, D. R. Reprogramming the specificity of sortase enzymes. Proc. Natl Acad. Sci. USA 111, 13343–13348 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Békés, M. et al. DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells. Cell Rep. 5, 826–838 (2013).

    Article  Google Scholar 

  34. 34.

    Husnjak, K. & Dikic, I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Kulathu, Y., Akutsu, M., Bremm, A., Hofmann, K. & Komander, D. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nat. Struct. Mol. Biol. 16, 1328–1330 (2009).

    CAS  Article  Google Scholar 

  36. 36.

    Zhang, X. et al. An interaction landscape of ubiquitin signaling. Mol. Cell 65, 941–955.e8 (2017).

    CAS  Article  Google Scholar 

  37. 37.

    Varadan, R., Assfalg, M., Raasi, S., Pickart, C. & Fushman, D. Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. Mol. Cell 18, 687–698 (2005).

    CAS  Article  Google Scholar 

  38. 38.

    Sims, J. J. & Cohen, R. E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference ofrap80. Mol. Cell 33, 775–783 (2009).

    CAS  Article  Google Scholar 

  39. 39.

    Sato, Y. et al. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80. EMBO J. 28, 2461–2468 (2009).

    CAS  Article  Google Scholar 

  40. 40.

    Bravo, R., Frank, R., Blundell, P. A. & Macdonald-Bravo, H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 326, 515–517 (1987).

    CAS  Article  Google Scholar 

  41. 41.

    Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    CAS  Article  Google Scholar 

  42. 42.

    Flotho, A. & Melchior, F. Sumoylation: a regulatory protein modification in health and disease. Annu. Rev. Biochem. 82, 357–385 (2013).

    CAS  Article  Google Scholar 

  43. 43.

    Pawale, V. S., Yadav, P. & Roy, R. P. Facile one-step assembly of bona fide SUMO conjugates by chemoenzymatic ligation. Chembiochem 19, 1137–1141 (2018).

    CAS  Article  Google Scholar 

  44. 44.

    Suree, N. et al. The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized. J. Biol. Chem. 284, 24465–24477 (2009).

    CAS  Article  Google Scholar 

  45. 45.

    Hirakawa, H., Ishikawa, S. & Nagamune, T. Design of Ca2+-independent Staphylococcus aureus sortase A mutants. Biotechnol. Bioeng. 109, 2955–2961 (2012).

    CAS  Article  Google Scholar 

  46. 46.

    Keren-Kaplan, T. et al. Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria. EMBO J. 31, 378–390 (2012).

    CAS  Article  Google Scholar 

  47. 47.

    Martinez-Fonts, K. & Matouschek, A. A rapid and versatile method for generating proteins with defined ubiquitin chains. Biochemistry 55, 1898–1908 (2016).

    CAS  Article  Google Scholar 

  48. 48.

    Nguyen, T. A., Cigler, M. & Lang, K. Expanding the genetic code to study protein-protein interactions. Angew. Chem. Int. Ed. Egnl. 57, 14350–14361 (2018).

    CAS  Article  Google Scholar 

  49. 49.

    Cigler, M. et al. Proximity-triggered covalent stabilization of low-affinity protein complexes in vitro and in vivo. Angew. Chem. Int. Ed. Engl. 56, 15737–15741 (2017).

  50. 50.

    David, Y., Vila-Perelló, M., Verma, S. & Muir, T. W. Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nat. Chem. 7, 394–402 (2015).

    CAS  Article  Google Scholar 

  51. 51.

    Blizzard, R. J. et al. Ideal bioorthogonal reactions using a site-specifically encoded tetrazine amino acid. J. Am. Chem. Soc. 137, 10044–10047 (2015).

    CAS  Article  Google Scholar 

  52. 52.

    Pickart, C. M. & Raasi, S. Ubiquitin and protein degradation, part B: controlled synthesis of polyubiquitin chains. in Methods in Enzymology, Vol. 399 (ed. Deshaies, R. J.) 26–28 (Elsevier, 2005).

  53. 53.

    Berndsen, C. E. & Wolberger, C. A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins. Anal. Biochem. 418, 102–110 (2011).

    CAS  Article  Google Scholar 

  54. 54.

    Bremm, A., Freund, S. M. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).

    CAS  Article  Google Scholar 

  55. 55.

    Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    CAS  Article  Google Scholar 

  56. 56.

    Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. J. Chem. Theory. Comput. 8, 3257–3273 (2012).

    CAS  Article  Google Scholar 

  57. 57.

    Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    Article  Google Scholar 

  58. 58.

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–28 (1996).

    Article  Google Scholar 

  59. 59.

    Schmied, W. H., Elsässer, S. J., Uttamapinant, C. & Chin, J. W. Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577–15583 (2014).

    CAS  Article  Google Scholar 

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This work was supported by the Excellence Initiative CIPSM and the DFG through the following programmes: GRK1721, SFB1309 and SPP1623 (to K.L.) as well as SFB1035 project B12 to V.R.I.K. and B10 to K.L. K.L. is a Mössbauer Professor at TUM-IAS. We thank C. Biertümpfel, MPI Martinsried for PCNA plasmids. Natively ubiquitylated PCNA was a generous gift from C. Biertümpfel. We thank M. Vermeulen, Radboud Insitute for Molecular Life sciences for GST-TAB2-NZF plasmid.

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K.L. conceived the research plan and experimental strategy. M.F. synthesized UAAs, performed all experiments in bacteria, including cloning, expression, purification of proteins and ubiquitylation/SUMOylation assays, as well as enzymatic assays and pull-down assays. A.-D.B. created PylRS libraries and evolved AzGGKRS. D.H.-G. performed initial mammalian cell experiments, including site-specific incorporation of AzGGK into HEK293T cells, and V.B. and A.B. performed ubiquitylation and SUMOylation assays in live HEK293T cells. A.J. and V.R.I.K. performed MD simulations. All authors analyzed data, and K.L. wrote the paper with input from the other authors.

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Correspondence to Kathrin Lang.

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We have filed a patent concerning the sortylation approach.

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Fottner, M., Brunner, AD., Bittl, V. et al. Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase. Nat Chem Biol 15, 276–284 (2019).

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