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Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins

Abstract

Enzymes are powerful tools for protein labelling due to their specificity and mild reaction conditions. Many protocols, however, are restricted to modifications at protein termini, rely on non-peptidic metabolites or require large recognition domains. Here we report a chemoenzymatic method, which we call lysine acylation using conjugating enzymes (LACE), to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9, and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. We demonstrate the utility of LACE by the site-specific attachment of biochemical probes, one-pot dual-labelling in combination with sortase, and the conjugation of wild-type ubiquitin and ISG15 to recombinant proteins.

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Fig. 1: Peptide thioesters as acyl donors for LACE.
Fig. 2: Isopeptide labelling of proteins using LACE.
Fig. 3: Reaction profile of LACE.
Fig. 4: Site-specific homo- and hetero-bifunctionalization.
Fig. 5: Application of LACE to one-step monoubiquitination and ISG15ylation of recombinant proteins.
Fig. 6: Structural investigations.

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Data availability

The X-ray structure of Ubc9 in complex with the isopeptide ligand has been deposited in the Protein Data Bank with accession code 6SYF. The following plasmids have been deposited in Addgene: pET28-His6-Ubc9, plasmid #133909; pET28-His6-Ubc9-C93A, plasmid #133910; pET28-His6-Ubc9-C138A, plasmid #133911; pET28-His6-GFP-C-LACE, plasmid #133913. Publicly available datasets used in this study can be accessed from the Protein Data Bank (PDB IDs 5F6E and 5D2M) and UniProt (entries P61956, P63279, Q8WZ42, P37840, P46060, P25963, P29590, P05161). All relevant data are included within the main text, Supplementary Information and source data files, and are available from the authors upon reasonable request. Source data are provided with this paper.

References

  1. Antos, J. M., Truttmann, M. C. & Ploegh, H. L. Recent advances in sortase-catalyzed ligation methodology. Curr. Opin. Struct. Biol. 38, 111–118 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Nguyen, G. K. T. et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732–738 (2014).

    CAS  PubMed  Google Scholar 

  3. Chang, T. K., Jackson, D. Y., Burnier, J. P. & Wells, J. A. Subtiligase: a tool for semisynthesis of proteins. Proc. Natl Acad. Sci. USA 91, 12544–12548 (1994).

    CAS  PubMed  Google Scholar 

  4. Shah, N. H. & Muir, T. W. Inteins: nature’s gift to protein chemists. Chem. Sci. 5, 446–461 (2013).

    PubMed Central  Google Scholar 

  5. Lin, C.-W. & Ting, A. Y. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc. 128, 4542–4543 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).

    CAS  PubMed  Google Scholar 

  7. Bellucci, J. J., Bhattacharyya, J. & Chilkoti, A. A noncanonical function of sortase enables site‐specific conjugation of small molecules to lysine residues in proteins. Angew. Chem. Int. Ed. 54, 441–445 (2015).

    CAS  Google Scholar 

  8. McConnell, S. A. et al. Protein labeling via a specific lysine–isopeptide bond using the pilin polymerizing sortase from corynebacterium diphtheriae. J. Am. Chem. Soc. 140, 8420–8423 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Fierer, J. O., Veggiani, G. & Howarth, M. SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture. Proc. Natl Acad. Sci. USA 111, E1176–E1181 (2014).

    CAS  PubMed  Google Scholar 

  10. Buldun, C. M., Jean, J., Bedford, M. R. & Howarth, M. SnoopLigase catalyzes peptide–peptide locking and enables solid-phase conjugate isolation. J. Am. Chem. Soc. 140, 3008–3018 (2018).

    CAS  PubMed  Google Scholar 

  11. Siegmund, V. et al. Spontaneous isopeptide bond formation as a powerful tool for engineering site-specific antibody–drug conjugates. Sci. Rep. 6, 39291 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Rabuka, D. Chemoenzymatic methods for site-specific protein modification. Curr. Opin. Chem. Biol. 14, 790–796 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2, 99–104 (2005).

    CAS  PubMed  Google Scholar 

  14. Puthenveetil, S., Liu, D. S., White, K. A., Thompson, S. & Ting, A. Y. Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J. Am. Chem. Soc. 131, 16430–16438 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815–15820 (2005).

    CAS  PubMed  Google Scholar 

  16. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell. Dev. 22, 159–180 (2006).

    CAS  Google Scholar 

  17. Chang, L. H. & Strieter, E. R. Reprogramming a deubiquitinase into a transamidase. ACS Chem. Biol. 13, 2808–2818 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Park, S., Krist, D. T. & Statsyuk, A. V. Protein ubiquitination and formation of polyubiquitin chains without ATP, E1 and E2 enzymes. Chem. Sci. 6, 1770–1779 (2014).

    PubMed  PubMed Central  Google Scholar 

  19. Johnson, E. S. Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382 (2004).

    CAS  PubMed  Google Scholar 

  20. Rodriguez, M. S., Dargemont, C. & Hay, R. T. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659 (2001).

    CAS  PubMed  Google Scholar 

  21. Hendriks, I. A. et al. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation. Nat. Struct. Mol. Biol. 24, 325–336 (2017).

    CAS  PubMed  Google Scholar 

  22. Bernier-Villamor, V., Sampson, D. A., Matunis, M. J. & Lima, C. D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108, 345–356 (2002).

    CAS  PubMed  Google Scholar 

  23. Stewart, M. D., Ritterhoff, T., Klevit, R. E. & Brzovic, P. S. E2 enzymes: more than just middle men. Cell Res. 26, 423–440 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhao, B. et al. SUMO‐mimicking peptides inhibiting protein SUMOylation. Chembiochem 15, 2662–2666 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhao, B. et al. Inhibiting the protein ubiquitination cascade by ubiquitin-mimicking short peptides. Org. Lett. 14, 5760–5763 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Harmand, T. et al. One-pot dual labeling of an IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1. Bioconjugate Chem. 29, 3245–3249 (2018).

    CAS  Google Scholar 

  27. 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  PubMed  Google Scholar 

  28. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    CAS  PubMed  Google Scholar 

  29. Udeshi, N. D. et al. Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition. Mol. Cell. Proteomics 11, 148–159 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–240 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Tatham, M. H. et al. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat. Cell Biol. 10, 538–546 (2008).

    CAS  PubMed  Google Scholar 

  32. Klug, H. et al. Ubc9 SUMOylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae. Mol. Cell 50, 625–636 (2013).

    CAS  PubMed  Google Scholar 

  33. Capili, A. D. & Lima, C. D. Structure and analysis of a complex between SUMO and Ubc9 illustrates features of a conserved E2–Ubl interaction. J. Mol. Biol. 369, 608–618 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Haj-Yahya, M. et al. Synthetic polyubiquitinated α-synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology. Proc. Natl Acad. Sci. USA 110, 17726–17731 (2013).

    CAS  PubMed  Google Scholar 

  35. Hejjaoui, M., Haj-Yahya, M., Kumar, K. S. A., Brik, A. & Lashuel, H. A. Towards elucidation of the role of ubiquitination in the pathogenesis of Parkinson’s disease with semisynthetic ubiquitinated α-synuclein. Angew. Chem. Int. Ed. 50, 405–409 (2011).

    CAS  Google Scholar 

  36. Rott, R. et al. SUMOylation and ubiquitination reciprocally regulate α-synuclein degradation and pathological aggregation. Proc. Natl Acad. Sci. USA 114, 13176–13181 (2017).

    CAS  PubMed  Google Scholar 

  37. Anderson, J. P. et al. Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739–29752 (2006).

    CAS  PubMed  Google Scholar 

  38. Hasegawa, M. et al. Phosphorylated α-synuclein is ubiquitinated in α-synucleinopathy lesions. J. Biol. Chem. 277, 49071–49076 (2002).

    CAS  PubMed  Google Scholar 

  39. Nonaka, T., Iwatsubo, T. & Hasegawa, M. Ubiquitination of α-synuclein. Biochemistry 44, 361–368 (2005).

    CAS  PubMed  Google Scholar 

  40. Lee, J. T., Wheeler, T. C., Li, L. & Chin, L.-S. Ubiquitination of α-synuclein by Siah-1 promotes α-synuclein aggregation and apoptotic cell death. Hum. Mol. Genet. 17, 906–917 (2007).

    PubMed  Google Scholar 

  41. Na, C. H. et al. Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J. Proteome Res. 11, 4722–4732 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Meier, F. et al. Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals differential effects on aggregation. J. Am. Chem. Soc. 134, 5468–5471 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Dzimianski, J. V., Scholte, F. E. M., Bergeron, É. & Pegan, S. D. ISG15: it’s complicated. J. Mol. Biol. 431, 4203–4216 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Theile, C. S. et al. Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1800–1807 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. Schulman, B. A. & Harper, J. W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 10, 319–331 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  47. McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G. & Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 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  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  50. Denuc, A. & Marfany, G. SUMO and ubiquitin paths converge. Biochem. Soc. Trans. 38, 34–39 (2010).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by ETH Zürich. We thank M. Levasseur for a gift of cell lines and support with flow cytometry; P. Mittl for help with X-ray analysis, and the team of the Swiss Light Source for technical assistance; S.-M. Duke for help with T4L labelling experiments; S. Shimura for synthetic rhodamine-labelled SUMO3; J. Farnung and C. J. White for helpful discussions; and the mass spectrometry service of the Laboratorium für Organische Chemie at ETH Zürich and the Functional Genomics Center Zürich for mass spectrometry analyses. G.A. acknowledges the Nakajima Foundation Scholarship. R.H. acknowledges the Stipendienfonds der Schweizerischen Chemischen Industrie (SSCI).

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Contributions

R.H., T.G.W. and J.W.B. conceived the project. R.H. designed and performed experiments, prepared materials and analysed data. G.A. designed and performed ubiquitination experiments, prepared materials and analysed data. T.G.W. performed initial experiments. R.H. and C.Z. performed structure determination by protein X-ray crystallography. J.W.B. designed experiments and analysed data. R.H. and J.W.B. wrote the manuscript with help from all authors.

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Correspondence to Jeffrey W. Bode.

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J.W.B., R.H. and T.G.W. are co-inventors on a European patent application which incorporates discoveries described in this manuscript.

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Extended data

Extended Data Fig. 1 Methods for internal protein labeling using short peptide tags.

Existing chemical (a) and chemoenzymatic methods (b) are summarized, together with the LACE method (c). Recognition sequence, as well as advantages and disadvantages of each method are listed. Parts of reagents and metabolites that can be altered to incorporate functional moieties are highlighted in red or shown as a red sphere.

Extended Data Fig. 2 The ubiquitination pathway and SUMOylation.

a, Mechanism of ubiquitin-like protein (Ubl) conjugation. Attachment of a Ubl to a substrate is initiated by an activating enzyme (E1) in an ATP-dependent process, forming a Ubl~E1 thioester intermediate. The Ubl is then transferred to a conjugating enzyme (E2) via transthioesterification, and lastly attached to the acceptor lysine of a target protein. The last step is often assisted by specific E3 ligases, for example RING type E3s (depicted), which bring together the Ubl~E2 thioester intermediate and a given substrate, contributing to target specificity and high reactivity. Deubiquitinases (DUBs) can hydrolyse the isopeptide bond. b, SUMOylation of RanGAP1 with rhodamine-labeled SUMO3 using E1, ATP and Ubc9, in the absence of an E3 ligase. c, In-gel fluorescence of SDS–PAGE analysis of b after 1 h reaction time in the presence (filled circle) or absence (hollow circle) of ATP. n = 2 independent experiments with similar results, representative data shown. Full gel image is available in the Source Data file.

Source data

Extended Data Fig. 3 Characterization of dual-modified trastuzumab Fab.

a, Structure of the sortase-reactive coumarin-glycine probe 20. b, Deconvoluted ESI-MS of unmodified (left) and modified (right) Fab after treatment of the sample with DTT to reduce the interchain disulfide bond (calc., calculated; obs, observed). Signals correspond to the light chain (LC, shown in grey), heavy chain (HC, shown in dark grey), and the coumarin- and rhodamine-modified products (light blue and red, respectively). c, Reducing SDS–PAGE analysis of the reaction over time, visualized by in-gel fluorescence to detect the presence of rhodamine from thioester 4 and coumarin from sortase probe 20, respectively, and by Coomassie stain. n = 3 independent experiments with similar results, representative data shown. Full gel images are available in the Source Data file.

Source data

Extended Data Fig. 4 Difference electron density of ligand and electrostatic interactions with Ubc9.

a, Difference electron density Fo–Fc after molecular replacement, contoured at 2.5 σ in red mesh. b, Ubc9-isopeptide structure without (top) or with (bottom) coulombic surface representation of the isopeptide ligand.

Supplementary information

Supplementary Information

Supplementary Figs. 1–37, Tables 1–3, general information and procedures, methods, NMR data and refs. 51–74.

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Supplementary Data

Data tables for Supplementary Figs. 3–6.

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Hofmann, R., Akimoto, G., Wucherpfennig, T.G. et al. Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins. Nat. Chem. 12, 1008–1015 (2020). https://doi.org/10.1038/s41557-020-0528-y

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