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. 2022 Feb 23;8(2):275-281.
doi: 10.1021/acscentsci.1c01490. Epub 2022 Feb 9.

Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9

Gaku Akimoto  1 , Arianna P Fernandes  1 , Jeffrey W Bode  1
Affiliations

Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9

Gaku Akimoto et al. ACS Cent Sci. .

Abstract

Ubiquitylation-the attachment of ubiquitin (Ub) to proteins in eukaryotic cells-involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics-due to a rate-limiting thioester loading step-and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant (k cat/K M) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono- and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Strategies for site-selective ubiquitylation using SUMO conjugating enzyme Ubc9. Ubc9–Ub thioester transfers Ub to the lysine residue in the LACE tag sequence, IKxE (x is any amino acid). (a) Lysine acylation using conjugating enzymes (LACE) using Ub–Mes thioester as an acyl donor. (b) Native Ub monomers or oligomers activated by an engineered E1 as an acyl donor.
Figure 2
Figure 2
Design and directed evolution of chimeric E1. (a) Structure of the spUba1–spUbc4 complex (PDB ID 4ii2) and Sae1/Sae2 heterodimer (PDB ID 1y8r). The E1s are shown in a surface model, and Ubc4 is shown in a ribbon model. Catalytic cysteine residues are highlighted in yellow. (b) Design of the chimeric E1, in which the UFD of Uba1 is replaced by that of Sae2. The interaction between SCCH and Ubc9 was further enhanced by directed evolution. The protein representation is based on Uba1 (PDB ID 6dc6). (c) Domain architecture of Uba1 ΔUFD, chimeric E1 (ChE) v0.1 and v0.2, in comparison to Sae1/Sae2 and Uba1. For detailed linking positions, see the Supporting Information. (d) E2 thioester formation assay using the E1 variants in part c. The reactions contained 0.2 μM E1, 5 μM Ubc9 Y134A, 5 μM biotin–Ub (Ubbt), 1 mM DTT, 5 mM ATP-Mg, 50 mM HEPES pH 7.5, 50 mM KCl and were incubated at 37 °C for 1 h. The reactions were resolved by SDS-PAGE and visualized by Western blot using streptavidin-HRP. (e) β-Lactamase exporting assay of representative chimeric E1 variants from each round of the directed evolution. (f) Locations of the 7 mutations in ChE1 v4.5. The protein representation is based on Uba1 (PDB ID 6dc6). (g) E2 thioester formation assay using Uba1, ChE1 v0.2 and v4.5, and reversion mutants of ChE1 v4.5. Conversion to Ubc9–Ub thioester was quantified by gel densitometry using Stain-Free imaging (BioRad). The reactions contained 0.5 μM E1, 3 μM Ubc9 K14R, 30 μM Ub, 0.5 U/mL inorganic pyrophosphatase, 0.5 mM DTT, 5 mM ATP-Mg, 50 mM HEPES pH 7.5, and 50 mM KCl and were incubated at 37 °C for 4 min. We could not test S748A since the mutant could not be expressed in E. coli. Data are presented as average values ± s.d. n = 3 independent experiments; individual data points are shown.
Figure 3
Figure 3
In vitro ubiquitylation. (a) Monoubiquitylation of GFP-I mediated by chimeric E1 and Ubc9. The LACE tag sequence is shown, in which the hydrophobic residue Ψ (bold), acceptor lysine K (blue), and acidic residue E (red) are highlighted. (b) Coomassie-stained SDS-PAGE of the reaction time-course and control reactions under the standard conditions using the acceptor lysine mutant K6R and omitting ChE1 v4.5 or Ubc9. (c) Quantification of the reaction conversion over time by gel densitometry using ChE1 v4.5 and v0.2 and Uba1. The apparent reaction half-time (t1/2) for ChE1 v4.5 is given. n = 2 independent experiments; individual data points are shown. (d) PolyUb transfer mediated by chimeric E1 and Ubc9. Linkage-defined Ub oligomers are prepared by using canonical E1, E2s, and DUBs (K11, K48, and K63) or by bacterial expression (M1) as previously reported. Transfer of Ub dimer (e) or trimer (f) to GFP-I under the standard conditions. (g) In-gel GFP fluorescence of transfer of K63 Ub hexamer to GFP-I under modified conditions (7.5 μM GFP-I, 0.5 μM ChE1 v4.5, 15 μM His6-Ubc9 K14R, 10 μM K63 Ub hexamer). Samples were not boiled to prevent smeared bands of polyUb, resulting in multiple bands of GFP and the different mobility of GFP variants. For a corresponding Coomassie-stained gel, see Figure S4. (h) Transfer of K63 Ub dimer to SUMO2 dimer.
Figure 4
Figure 4
Monoubiquitylation in E. coli. (a) Constructs coding for MBP-substrate (pSub), His6-Ub, Ubc9, and chimeric E1 v4.5 (pUb). Monoubiquitylation and purification of GFP-I (b) and of HRas (c). Deconvoluted ESI-MS of the products (calc., calculated; obs., observed) and Coomassie-stained SDS-PAGE are shown. HRas–Ub is partly glycosylated (+162 Da). Protein bands marked with an asterisk are TEV protease. The predicted structure of HRas was obtained from the AlphaFold Protein Structure Database. A detailed purification scheme and the complete SDS-PAGE and anti-Ub Western blot images, including GFP-I tag K6R and HRas K170R mutants, are available in Figure S5.
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