Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1

Abstract

Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages and lengths. Notably, this strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. Our work not only deepens the understanding of how an E3-free substrate ubiquitination reaction occurs in human cells, but also provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination.

Fig. 1. Structure of the E2 enzyme UBE2E1 in complex with a SETDB1-derived peptide.

a In vitro UBE2E1-dependent ubiquitination assay using hexapeptide (KEGYES)-fused EGFP as substrates. Gel images are representative of independent biological replicates (n = 2). b Mass spectrometry confirms the ubiquitination product (EGFP*-Ub) and the lysine on the hexapeptide is the ubiquitination site. The observed (obs.) and calculated (calc.) molecular weights were marked. c Overall structure of UBE2E1 bound to hexapeptide. UBE2E1 is shown as ribbon and colored in bright blue. The peptide is shown as sticks: the long side, corner, and the short side of the “L shape”-hexapeptide are colored in bright orange, green and cyan, respectively. Source data are provided as a Source Data file.

Fig. 2. Molecular insights into UBE2E1-mediated ubiquitination without E3.

a Detailed interactions of hexapeptide with UBE2E1 residues. The intermolecular hydrogen bonds are indicated with yellow dashed and the residues are shown as sticks with the same color code as in Fig. 1c. b UBE2E1 D136 residue poised to activate the modeled lysine residue’s nucleophilicity. c Interaction diagram between UBE2E1 and SEDTB1-derived peptide. Intermolecular hydrogen bonds and hydrophobic interactions are indicated with yellow dashes and arrows, respectively. d The proper distance between the receptor lysine and glycine at the corner of “L-shaped” hexapeptide is necessary for ubiquitination. e Engineered Ubch5c enabled site-specific ubiquitination in an E3-free manner. Gel images shown in (d) and (e) are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.

Fig. 3. Exploring sequence-dependent ubiquitination by UBE2E1 (SUE1) as a general strategy for protein ubiquitination.

a Schematic representation of Uba1/UBE2E1-mediated site-specific ubiquitination, in which the protein of interest (POI) bears the SUE1 tag (KEGYEE) at the customized site and monoUb or ubiquitin chain is used as the ubiquitin source. Generally, 1 μM Uba1, 20 μM UBE2E1, 8 μM substrate containing the SUE1 tag, 80 μM monoUb or 40 μM Ub chain were mixed, reacted in the reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂ and 10 mM ATP) at 37 °C for 2 h. b The SUE1 strategy for ubiquitination at different sites, the N-terminus, internal region or C-terminus of EGFP was chosen to introduce a SUE1 tag, respectively. c The SUE1 strategy for ubiquitination with different linkages of Ub chains, M1/K6/K11/K27/K29/K33/K48/K63-linked diUb was used to transfer to the substrate respectively. d The SUE1 strategy for ubiquitination with different lengths of Ub chains, taking K48-linked Ub chains as an example. e The SUE1 strategy for ubiquitination with branched Ub chain (K11/K48 branched). Gel images shown in (b–e) are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.

Fig. 4. Ubiquitination of authentic substrate protein via the SUE1 strategy.

a Top, SDS‒PAGE analysis of SUE1-mediated monoubiquitination of α-Synuclein (α-Syn) at K43 using In-gel fluorescence for clear visualization of ubiquitination. The model of α-Synuclein was derived from the NMR structure of the wild-type α-Synuclein (PDB: 1XQ8). Bottom, deconvoluted ESI-MS of the purified monoubiquitinated α-Synuclein (α-Syn-Ub). b Top, SDS‒PAGE analysis of SUE1-Mediated K63-linked ubiquitin chain modification of p53 at K24 and asterisk (*) denotes an impurity in the p53-stock. The p53 model was derived from the predicted structure of wild-type p53 in the AlphaFold Protein Structure Database (AF-P04637-F1-model_v4). Bottom, deconvoluted ESI-MS of the purified diubiquitinated p53 (p53-diUb). c Top, SDS‒PAGE analysis of SUE1-mediated monoubiquitination of p53 at K386 and asterisk (*) denotes an impurity in the p53-stock. Deconvoluted ESI-MS of the purified ubiquitinated products. Bottom, deconvoluted ESI-MS of the purified monoubiquitinated p53 (p53-Ub). Gel images shown in (a–c) are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.

Fig. 5. Application of SUE1 to defined multisite ubiquitinated proteins and site-specific NEDD8-modified proteins.

a Schematic representation of SUE1-mediated multisite ubiquitination, in which protein of interesting (POI) bears multiple (here two) SUE1 tags (KEGYEE) at customized sites. Generally, 1 μM Uba1, 20 μM UBE2E1, 8 μM substrate containing multiple SUE1 tags, 40 μM monoUb or Ub chain were mixed and reacted in the reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂ and 10 mM ATP) at 37 °C for 2 h. b SDS‒PAGE analysis of SUE1 mediated dual-site monoubiquitination at K43 and K96 of α-synuclein (α-Syn). c Schematic representation of the SUE1 strategy in combination with the LACE strategy for multi-site ubiquitinated proteins with different Ub chain linkages, in which protein of interesting (POI) bears the SUE1 tag and LACE tag at customized sites. d SDS–PAGE analysis of SUE1 and LACE co-mediated dual-site ubiquitination of α-synuclein (α-Syn) and K48-linked diUb modification at K43 by SUE1 strategy and monoubiquitination at K96 by LACE strategy. In general, 1 μM Uba1, 40 μM UBE2E1, 16 μM substrate containing one SUE1 tag and one LACE tag and 16 μM Ub chain (or monoUb) were mixed and reacted in reaction buffer to achieve almost complete ubiquitination of substrate, and then 1 μM chimera E1 V4.5, 40 μM UBC9 K14R and 160 μM another Ub chain (or monoUb) were added to the reaction buffer at 37 °C for another 2 h. e Schematic representation of Uba1/UBE2E1-mediated site-specific neddylation, in which protein of interesting (POI) bears the SUE1 tag (KEGYEE) at the customized site. The reaction conditions were the same as for mono-ubiquitination, except NEDD8 (N8) was used to replace monoUb. f SDS‒PAGE analysis of SUE1-Mediated NEDD8-modification of EGFP. g deconvoluted ESI-MS of the purified neddylated EGFP (EGFP-N8). Partially EGFP was gluconoylated on His tag (+178 Da) during expression in E. coli. Gel images shown in (b), (d) and (f) are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.

Fig. 6. SUE1 facilitates biochemical evaluation of proteasomal degradation signals.

a Schematic illustration of the route used to generate K48-linked or K29/48-branched pentaUb modified NCB1 for degradation assays. b In-gel-fluorescence analysis of ubiquitinated NCB1 and Coomassie-stained SDS‒PAGE gel can be found in Supplementary Fig. 10. c Ubiquitinated NCB1 was mixed with purified 26S proteasomes for indicated time periods and degradation of substrates was detected by In-gel-fluorescence. d The average percentage of residual Ubiquitinated NCB1 at indicated time points after degradation (from c). Data represent the mean ± SD of three independent experiments. Gel images in (b) represent independent biological replicates (n = 2), and in (c) represent independent biological replicates (n = 3). Source data are provided as a Source Data file.