Development of D-box peptides to inhibit the anaphase-promoting complex/cyclosome
Figures

Structure and function of anaphase-promoting complex/cyclosome (APC/C).
(A) Schematic of APC/C activity during mitotic exit, indicating the switch in co-activator from Cdc20 to FZR1. Most substrates contain variable degrons (D-box in green, KEN in yellow) present in IDRs. (B) Domain structuring of Cdc20 comprising an N-terminal IDR with the C-box, KEN-box, and CRY-box motifs, the central WD40 domain responsible for substrate recruitment via the degron binding sites and the C-terminal IDR containing the IR-tail. (C) Schematic of the structure of the Cdc20 WD40 domain (PDB: 4GGC) overlaid with those of the WD40 domain in complex with Acm1 D-box and ABBA motif peptides (PDB: 4BH6) (He et al., 2013) and the KEN-box peptide 4GGD (Tian et al., 2012).

Biophysical characterisation of Apcin binding to Cdc20WD40 by thermal shift assay (TSA) and surface plasmon resonance (SPR).
(A) Representative examples of thermal unfolding traces of Cdc20 WD40 in the presence of 1% DMSO as the vehicle control or Apcin at concentrations of 25, 50, and 100 µM. (B) Corresponding melting temperatures calculated from derivative plots of the thermal unfolding traces. Mean data from triplicate measurements are shown, with error bars representing standard deviations. (C) Reference-subtracted sensorgrams of biotinylated Cdc20WD40 and Apcin. (D) Binding affinity determination of Apcin to Cdc20WD40 domain by steady-state analysis of the sensorgrams.
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Figure 2—source data 1
TSA data and BLI sensorgram data for Figure 2.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig2-data1-v1.xlsx

Verification of single-site biotinylation of purified Cdc20 WD40 domain (residues 161-477) using Sulfo-NHS-LC-LC-Biotin (Thermo Scientific, A35358).
The observed mass shift corresponds to one Sulfo-NHS-LC-LC-Biotin molecule added to the purified protein.

D-box peptide mutations.
(A) Schematic showing the Acm1 D-box peptide bound to yeast FZR1 homologue Cdh1. R119 of the D-box forms H-bond interactions with D256 and E537 of Cdh1. L122 of the D-box buries into the canonical pocket on the surface of Cdh1 (PDB: 4BH6, He et al., 2013). (B) Melting temperature of Cdc20WD40 in the presence of D-box peptides at 25, 50, and 100 µM concentrations, calculated from derivative plots of the thermal unfolding traces. Mean data from triplicate measurements are shown, with error bars representing standard deviations.
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Figure 3—source data 1
TSA melting temperature data for Figure 3B.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig3-data1-v1.xlsx

Reference-subtracted surface plasmon resonance (SPR) sensorgrams and binding curves for (A) D1, (B) D3, (C) D4, (D) D5, (E) D10, (F) D19 binding to Cdc20.

Linear amino acid sequence and chemical structures for peptides D1 and D2 are illustrated alongside the expected exact mass and molecular weights for each peptide.
LCMS data showing the m/z species for each purified peptide. Analytical HPLC chromatograms (10–30% solvent B over 15 minutes, 1 mL l/min, UV trace at 220 nm), with calculated purity of the peptides by the percentage area.

Linear amino acid sequence and chemical structures for peptides D3 and D4 are illustrated alongside the expected exact mass and molecular weights for each peptide.
LCMS data showing the m/z species for each purified peptide. Analytical HPLC chromatograms (10–30% solvent B over 15 minutes, 1 mL l/min, UV trace at 220 nm), with calculated purity of the peptides by the percentage area.

Linear amino acid sequence and chemical structures for peptides D5, D10 and D19 are illustrated alongside the expected exact mass and molecular weights for each peptide.
LCMS data showing the m/z species for each purified peptide. Analytical HPLC chromatograms (10–30% solvent B over 15 minutes, 1 mL l/min, UV trace at 220 nm), with calculated purity of the peptides by the percentage area.

D-box peptides incorporating unnatural amino acids.
(A) Schematics of the two unnatural amino acids used. (B) Thermal stabilisation of the Cdc20WD40 by the two highest affinity peptides D20 and D21 calculated from derivative plots in thermal shift assay (TSA) . Surface plasmon resonance (SPR) reference-subtracted sensorgrams and binding curves for (C) D20 and (D) D21.
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Figure 4—source data 1
BLI sensorgram data and TSA melting temperature data for Figure 4.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig4-data1-v1.xlsx

Linear amino acid sequence and chemical structures for peptides D7 and D12 are illustrated alongside the expected exact mass and molecular weights for each peptide.
LCMS data showing the m/z species for each purified peptide. Analytical HPLC chromatograms (10–30% solvent B over 15 minutes, 1 mL l/min, UV trace at 220 nm), with calculated purity of the peptides by the percentage area.

Linear amino acid sequence and chemical structures for peptides D20 and D21 are illustrated alongside the expected exact mass and molecular weights for each peptide.
LCMS data showing the m/z species for each purified peptide. Analytical HPLC chromatograms (10–30% solvent B over 15 minutes, 1 mL l/min, UV trace at 220 nm), with calculated purity of the peptides by the percentage area.

Crystal structures of Cdc20-D-box complexes.
X-ray crystal structures of peptides (A) D21, (B) D20, and (C) D7 bound to the canonical D-box binding pocket of Cdc20. Intermolecular hydrogen bonds between peptides and Cdc20 are shown by dashed lines. (D) Structural alignment of D21-bound Cdc20 and Acm1 D-box peptide bound to Cdh1 (PDB: 4BH6; He et al., 2013). Peptide backbones align with an RMSD of 1.007 Å. Modelled water molecules have been removed from images for clarity.

Cdc20WD40 is shown in cartoon (cyan) and surface (grey) representations.
All peptides are shown by stick representations (magenta). Intramolecular hydrogen bonds formed within peptides (A) D7, (B), D20, and (C) D21. All peptides form a hydrogen bond between the carbonyl of A2 to the amine of G5/S5. Peptides D20 and D21 form an additional H-bond between the carbonyl of A2 to the hydroxyl group of S5. 2FoFc maps contoured at 1.0σ and modelled peptide atoms of (D) D7, (E), D20, and (F) D21. Unbiased FoFc maps from refinement steps prior to including the peptides in the model, contoured at 2.5σ. Maps shown are (G) D7, (H) D20, and (I) D21.

D-box peptides bind to full-length HiBiT-tagged Cdc20 in the cellular context.
Representative cellular thermal shift assay (CETSA) data are shown for Cdc20-tranfected HEK293T cell lysates incubated with D-box peptides at a concentration of 100 µM.
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Figure 6—source data 1
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig6-data1-v1.xlsx

CETSA method development and validation using Apcin as a positive control.
(A) Cellular thermal shift assay (CETSA) of endogenous Cdc20 in HEK293T cell lysates by 100 µM Apcin compared with vehicle control (1% DMSO), analysed by densitometric analysis of western blots. Mean and standard deviation are calculated from two independent experiments. (B) CETSA of transfected Cdc20 with a C-terminal HiBiT tag spiked with 1% DMSO with and without including a centrifugation step following heat denaturation at each temperature set point. (C) Stabilisation of transfected Cdc20 with a C-terminal HiBiT tag by 100 µM Apcin compared with vehicle control (1% DMSO).
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Figure 6—figure supplement 1—source data 1
Original file from one experimental replicate for western blot analysis of a CETSA experiment displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original western blots labelled from the source data 1 used in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
Original file from one experimental replicate for western blot analysis of a CETSA experiment displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig6-figsupp1-data3-v1.zip
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Figure 6—figure supplement 1—source data 4
Original western blots labelled from source data 3 used in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig6-figsupp1-data4-v1.zip

Inhibition of APC/CCdc20-mediated ubiquitination of Cyclin B1 by D-box peptides and Apcin.
In vitro ubiquitination assays using reconstituted APC/CCdc20 with Cyclin B1 as the substrate for ubiquitination. Lead peptides and Apcin were titrated from 3 µM to 300 µM and showed concentration-dependent inhibition of Cyclin B1 ubiquitination compared to the vehicle control (0.7% DMSO).
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Figure 7—source data 1
Original western blots used in Figure 7.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig7-data1-v1.zip
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Figure 7—source data 2
Original western blots used in Figure 7.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig7-data2-v1.zip

D-box variants can drive degradation in mitotic cells.
(A) Schematic of D-box-mNeon constructs used in fluorescence timelapse imaging. (B) mNeon fluorescence levels in individual cells plotted over time to show D-box mediated degradation of mNeon in mitosis. Fluorescence measurements from individual cells are normalised to fluorescence at metaphase then in silico synchronised to anaphase onset. Mean degradation curves are shown, with error bars representing SDs. (C) Degradation rate curves show rate of change in relative fluorescence of the D-box variants and reveal maximum degradation rate for each construct. Error bars are depicted as shaded regions and indicate SDs. (D) Levels of relative fluorescence in each cell at t=1 hour after anaphase onset. Degradation of each D-box construct was significant relative to D0 control, using Welch’s t-test. ****p≤0.0001. In (B–D), n=D0 (20) D1 (23), D2 (40), D3 (38), and D19 (34), with data pooled from two or more independent experiments.
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Figure 8—source data 1
Data for the time dependence of mNeon fluorescence for Figure 8.
- https://cdn.elifesciences.org/articles/104238/elife-104238-fig8-data1-v1.xlsx
Tables
Binding of D-box peptides to Cdc20WD40 measured by surface plasmon resonance and thermal shift assay.
Peptide | Sequence | KD(µM) | ΔTm(°C at 100 µM peptide) |
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D1 | Ac-GRAALSDITN-NH2 | 18.6±0.2 | 1.9±0.1 |
D2 | Ac-RLPLGDISN-NH2 | n.d. | 0.3±0.3 |
D3 | Ac-RAPLGDVSN-NH2 | 54.4±0.7 | 1.7±0.3 |
D4 | Ac-RAPLGDISN-NH2 | 19.6±0.2 | 1.51±0.05 |
D5 | Ac-RAPLGDLSN-NH2 | 27±1 | 1.5±0.1 |
D10 | Ac-RAALGDISN-NH2 | 70±3 | 0.6±0.2 |
D19 | Ac-RAPLSDITN-NH2 | 5.9±0.1 | 3.4±0.1 |
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n.d. indicates not detectable.
Binding of D-box peptides containing unnatural amino acids replacing Leu4 binding to Cdc20WD40 measured by surface plasmon resonance and thermal shift assay.
Reported values are the mean ± standard deviation of triplicate measurements.
Peptide | Sequence | KD(µM) | ΔTm(°C, at 100 µM peptide) |
---|---|---|---|
D7 | Ac-RAPC3GDISN-NH2 | 3.1±0.1 | 4.13±0.03 |
D12 | Ac-RAAC3GDISN-NH2 | 13.3±0.1 | 2.0±0.3 |
D20 | Ac-RAPC3SDITN-NH2 | 0.90±0.01 | 6.3±0.1* |
D21 | Ac-RAPF3SDITN-NH2 | 0.52±0.01 | 6.7±0.1 |
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*
Standard deviation could not be determined, and the error was estimated based on uncertainties in peptide and protein concentrations.
Melting temperatures of HiBiT-tagged Cdc20 in the presence of 100 µM D-box peptides measured by cellular thermal shift assay.
Melting temperatures are calculated from the mean of three experiments, and the standard deviations are listed.
Sample | Melting temperature (°C) |
---|---|
DMSO only | 50.0±0.4 |
D19 | 50.4±0.6 |
D7 | 51.2±0.3 |
D20 | 52.6±0.4 |
D21 | 53.2±0.8 |
Additional files
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Supplementary file 1
Data collection, phasing, and refinement statistics for Cdc20 crystal structures with D-box peptides.
- https://cdn.elifesciences.org/articles/104238/elife-104238-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104238/elife-104238-mdarchecklist1-v1.docx