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.

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.

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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.

Source data

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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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