Research ArticleStructural Biology

Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S

See allHide authors and affiliations

Science Signaling  20 Oct 2020:
Vol. 13, Issue 654, eaba8208
DOI: 10.1126/scisignal.aba8208
  • Fig. 1 UBE2S self-associates in vitro and colocalizes in cells.

    (A) Representative PLA stains for HA and FLAG epitopes in HCT116 UBE2S−/− cells cotransfected with plasmids encoding HA-UBE2S and FLAG-UBE2S. As a negative control, HA-UBE2S was coexpressed with FLAG-Venus. As a positive control, HA-ubiquitin (Ub) was coexpressed with FLAG-UBE2S. As a technical control, HA-UBE2S was expressed by itself. Bright dots indicate the colocalization of FLAG- and HA-tagged molecules. Because of varying signal intensities, the images were individually adjusted. Scale bar, 10 μm. (B) Quantification of PLA signals as shown in (A). All images were treated identically. Black bars represent the median of two independent experiments. N indicates the number of analyzed cells. “Ratio” denotes the median signal normalized to that of the HA-UBE2S + FLAG-UBE2S experiment. Statistical significance was calculated according to Kruskal-Wallis and Dunn’s multiple comparison test. (C) Amide proton relaxation rates determined for UBE2SUBC and UBE2S at the indicated concentrations by NMR (N = 1). (D) Ni2+-NTA resin–based in vitro pull-down experiment with the indicated tagged UBE2S variants, monitored by SDS-PAGE and immunoblotting (N = 3 independent experiments). As negative controls, the resin was incubated with HA-UBE2S and His6-UBE2S only, respectively.

  • Fig. 2 Structure-guided cross-linking defines a specific dimerization mode of UBE2S in solution.

    (A) Superposition of the crystal structures of UBE2SUBC wild type (PDB, 6S98) and C118A (PDB, 6S96), determined here. Cys95 in 6S96 is oxidized to a sulfoxide. (B) Cartoon representation of the dimer [see (A)]. (C) SEC of UBE2SUBC wild type and variants (N = 3 independent experiments). (D) SEC analogous to (B) upon bBBr-based cross-linking (N = 2 independent experiments). (E) bBBr-based cross-linking of UBE2SUBC, monitored by SDS-PAGE (N = 3 independent experiments). (F) SEC MALS analysis of UBE2SUBC after incubation with or without bBBr. Determined MWs: 16.5 ± 0.3 kDa (monomer) and 31.7 ± 0.5 kDa (dimer) (N = 2 independent experiments). (G) SEC MALS analysis of UBE2S after incubation with or without bBBr (N = 2 independent experiments). Determined MWs: 26 ± 2 kDa (monomer) and 47 ± 2 kDa (dimer). Note that SEC MALS (F and G) required higher protein concentrations than SEC (C and D), producing a larger fraction of dimer. (H) bBBr-based cross-linking of UBE2S, monitored by SDS-PAGE (N = 3 independent experiments). (I) pKa determination for the thiol group of Cys95 in UBE2S C118S based on the reaction kinetics with DTNB. (J) pKa determination for the thiol group of Cys118 in UBE2S C95S, determined as in (I). In (I) and (J), the mean and SD are plotted (N = 5 independent experiments). (K) Immunoblot of mitotically enriched, bBBr-treated extract from RPE-1 cells stably expressing Tet-induced HA-UBE2S and transiently expressing FLAG-UBE2S, separated by SEC (N = 3 independent experiments). For the HA immunoblot, see fig. S2A. (L) Immunoblot of control immunoglobulin G (IgG) and anti-FLAG immunoprecipitations (IP) from the indicated, pooled SEC fractions from (K) (N = 2 independent experiments).

  • Fig. 3 UBE2S dimerization requires the hydrophobic face of helix αB and is promoted by the C-terminal extension.

    (A) Detail of the dimerization interface in the crystal structure of UBE2SUBC wild type (PDB, 6S98; see Fig. 2A); contacting side chains are illustrated as sticks. (B) Relative bBBr-based cross-linking rates of 26 purified UBE2SUBC and UBE2S variants. (C) Domain organization of UBE2S. (D) Relative bBBr-based cross-linking rates of three UBE2S variants of different length. (E) Competition experiment testing the effect of a 10-fold molar excess of a C-helix–derived peptide in trans on the bBBr-based cross-linking kinetics of UBE2S. (F) Relative bBBr-based cross-linking rates of UBE2S1-196 and a UBE2S1-197-Ub fusion protein. In (B) and (D to F), the mean and SD are plotted (N = 3 independent experiments); for raw data, see figs. S3B and S4 (A to C).

  • Fig. 4 Dimerization of UBE2S confers autoinhibition.

    (A) Crystal structure of UBE2SUBC with a “donor-like” ubiquitin molecule [the complex is formed in trans, but recapitulates the closed state; PDB, 5BNB (13)]. The side chains of Cys95, Leu107, His111, and Leu114 (which were selected for mutational studies) are shown. (B) Crystal structure of the UBE2SUBC dimer (PDB, 6S98; see Fig. 2A) with side chains displayed as in (A). (C) Comparison of the isopeptide bond formation activities of UBE2S and its cross-linked dimeric form (N = 3 independent experiments). The products of autoubiquitination (monomer-Ubn; highlighted by the red line) and unanchored ubiquitin chain formation (Ub2) are labeled. The asterisk denotes minor degradation of the dimer. (D) Comparison of the ubiquitin thioester formation activities of UBE2S and its cross-linked dimeric form (N = 3 independent experiments). Available thioester-linked products (monomer~Ub) are indicated and sensitive to reducing agent (DTT). Both (C) and (D) include control reactions without ATP.

  • Fig. 5 Disruption of the dimer interface reduces UBE2S colocalization in the cell.

    (A) Representative PLA and immunostains for HA and FLAG epitopes in HCT116 UBE2S−/− cells cotransfected with plasmids encoding the indicated HA/FLAG-tagged UBE2S variants. Because of varying signal intensities, the PLA images were individually adjusted. Scale bars, 10 μm. (B) Quantification of PLA signals. Black bars represent the median from three independent experiments, as shown in (A). N indicates the number of analyzed cells. Ratio denotes the median signal normalized to that of UBE2S WT. Statistical significance according to the Kruskal-Wallis and Dunn’s multiple comparison test (left) and an unpaired, two-tailed Mann-Whitney test (right), respectively. Note that we did not observe a correlation between the cellular amount of the HA- or FLAG-tagged UBE2S variants, respectively, and the number of PLA dots.

  • Fig. 6 Disruption of the dimer interface increases UBE2S turnover and prevents mitotic slippage.

    (A) Immunoblot establishing the siRNA-and-rescue system in RPE-1 cells replacing endogenous with Tet-inducible UBE2S, expressed from the same mRNA as GFP (N = 3 independent experiments). Cells were arrested in prometaphase by addition of DMA. Chromosome segregation 1 protein (CSE1) serves as a loading control. (B) Immunoblot monitoring UBE2S stability in the absence of translation. Cells treated as in (A) were complemented with UBE2S wild type and H111A, respectively, and exposed to cycloheximide (CHX) for the indicated times. To assess the contribution of proteasomal activity, MG132 was added, as indicated. (C) Fluorescence imaging–based quantification of UBE2S amounts [as monitored in (B)], normalized to GFP. The data represent the mean and SD (N = 3 independent experiments). (D) Half-lives (mean and SD) of the indicated UBE2S variants, derived from nonlinear fitting (one-phase decay) of the data in (B) (for wild type and H111A) and in fig. S9 (A and B) (for L107A and L114E). Significance according to Kruskal-Wallis and Dunn’s multiple comparison test. (E and F) Automated live-cell imaging-based quantification of the release of cells treated as in (A) and expressing UBE2S wild-type and H111A, respectively, from a DMA-induced SAC arrest. The data represent the mean and SD from nine measurements (N = 3 independent experiments), normalized to the number of mitotic cells at the start (t0). (G) Immunoblot showing the amount of UBE2S wild type and H111A, respectively, during a DMA-induced SAC arrest in cells treated as in (A). (H) Quantification of the relative amount of UBE2S H111A (normalized to the WT amount) in prometaphase, normalized to GFP; mean and SD of data as shown in (G) (N = 3 independent experiments). A WT bar is illustrated as a reference only. (I) Analysis of mitotic slippage of cells as in (A), but exposed to Taxol, based on live-cell imaging and single-cell analysis. The duration of the mitotic arrest was measured on the basis of 202 cells per condition (N = 2 independent experiments). Black lines indicate the median. Red dots reflect cells that did not complete mitosis during the experiment.

  • Fig. 7 Model of the conformational regulation of UBE2S, as analyzed in this study.

    The catalytic UBC domain of UBE2S can form an autoinhibited dimer, which is stabilized by the C-helix, possibly through intersubunit contacts or allosterically. The C-helix concomitantly anchors UBE2S at the APC2/4 groove during ubiquitin chain elongation on APC/C substrates (8), stimulates chain initiation by UBE2C (11), and provides major autoubiquitination sites that promote the proteasomal turnover of UBE2S (15, 16, 18, 39). Our model posits that the formation of the inhibited dimer stabilizes UBE2S in the cell by preventing autoubiquitination at the C-helix. Additional states in the conformational landscape of UBE2S not shown in this figure include the autoinhibited, Lys+5-ubiquitinated form (18), interactions between the C-helix and the APC/C coactivators (10, 15, 19), and the architecture of the APC/C during ubiquitin chain initiation (710).

  • Table 1 X-ray crystallographic data collection and refinement statistics.

    Values in parentheses correspond to the highest-resolution shell. WT, wild type.

    UBE2SUBC WTUBE2SUBC C118A
    (PDB, 6S98)(PDB, 6S96)
    Data collection
      Wavelength (Å)0.96801.033
      Space groupP 1 21 1P 61
      Unit cell parameters
        a b c (Å)44.8 49.05 71.93120.9 120.9 45.3
        α β γ (°)90 106.03 9090 90 120
      Total reflections78466 (7412)39533 (3800)
      Unique reflections43484 (4292)19810 (1898)
      Rpim2.5 (43.0)4.4 (40.8)
      Completeness (%)99.4 (98.9)98.8 (95.1)
      I/σ(I)14.5 (2.0)8.6 (1.5)
      Multiplicity1.8 (1.7)2.0 (2.0)
      Wilson B factor18.844.02
      CC ½0.999 (0.638)0.997 (0.992)
    Refinement
      Resolution (Å)42.15–1.55
    (1.605–1.55)
    34.91–2.18
    (2.258–2.18)
      Rwork/Rfree16.14/18.3920.38/24.53
      No. of atoms
        Protein23582241
        Water17132
      Average B-factors
        Protein29.557.2
        Water32.149.8
      RMSD from ideality
        Bond lengths (Å)0.0130.014
        Bond angles (°)1.351.21
      Ramachandran statistics
        Favored (%)98.9698.36
        Disallowed (%)0.000.00
      MolProbity clash score3.359.42
      MolProbity overall score1.221.5

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/654/eaba8208/DC1

    Fig. S1. Characterization of UBE2S colocalization and self-association.

    Fig. S2. Characterization of UBE2S cross-linking in cells and in vitro.

    Fig. S3. bBBr-based cross-linking kinetics of UBE2S dimer interface variants.

    Fig. S4. bBBr-based cross-linking kinetics of additional UBE2S variants.

    Fig. S5. Effect of the C-helix on the UBC domain of UBE2S.

    Fig. S6. Mass spectrometric analyses of bBBr-based UBE2S cross-linking.

    Fig. S7. NMR-based comparison of the interactions between UBE2S variants and ubiquitin.

    Fig. S8. Activity assays with UBE2S wild-type and dimer interface variants.

    Fig. S9. Characterization of UBE2S dimer interface variants in cells and in vitro.

    Table S1. Peak list for the deconvoluted mass spectrum shown in fig. S6A.

    Table S2. Mapping of bBBr-cross-linking sites in the UBE2S dimer by ESI-MS shown in fig. S6C.

    Table S3. Plasmids, primers, and cloning information.

    Table S4. Antibodies.

    References (72, 73)

  • This PDF file includes:

    • Fig. S1. Characterization of UBE2S colocalization and self-association.
    • Fig. S2. Characterization of UBE2S cross-linking in cells and in vitro.
    • Fig. S3. bBBr-based cross-linking kinetics of UBE2S dimer interface variants.
    • Fig. S4. bBBr-based cross-linking kinetics of additional UBE2S variants.
    • Fig. S5. Effect of the C-helix on the UBC domain of UBE2S.
    • Fig. S6. Mass spectrometric analyses of bBBr-based UBE2S cross-linking.
    • Fig. S7. NMR-based comparison of the interactions between UBE2S variants and ubiquitin.
    • Fig. S8. Activity assays with UBE2S wild-type and dimer interface variants.
    • Fig. S9. Characterization of UBE2S dimer interface variants in cells and in vitro.
    • Table S1. Peak list for the deconvoluted mass spectrum shown in fig. S6A.
    • Table S2. Mapping of bBBr-cross-linking sites in the UBE2S dimer by ESI-MS shown in fig. S6C.
    • Table S3. Plasmids, primers, and cloning information.
    • Table S4. Antibodies.
    • References (72, 73)

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article