Structure and Function of 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 TSA and 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.

Binding of D-box peptides to Cdc20WD40 measured by SPR and TSA.

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.

Binding of D-box peptides containing unnatural amino acids replacing Leu4 binding to Cdc20WD40 measured by SPR and TSA. Reported values are the mean ± standard deviation of triplicate measurements.

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 TSA. SPR reference-subtracted sensorgrams and binding curves for (C) D20, and (D) D21.

Crystal structures of Cdc20-D-box complexes.

X-ray crystal structures of peptides (A) D21, (B) D20 & (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 to with an RMSD of 1.007Å. Modelled water molecules have been removed from images for clarity.

Melting temperatures of HiBiT-tagged Cdc20 in the presence of 100 μM D-box peptides measured by CETSA. Melting temperatures are calculated from the mean of three experiments, and the standard deviations are listed.

D-box peptides bind to full-length HiBiT-tagged Cdc20 in the cellular context.

Representative CETSA data are shown for Cdc20-tranfected HEK293T cell lysates incubated with D-box peptides at a concentration of 100 µM.

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 300 µM to 3 µM and showed concentration-dependent inhibition of Cyclin B1 ubiquitination compared to the vehicle control (0.7% DMSO).

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 normalized to fluorescence at metaphase then in silico synchronized 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), D19 (34) with data pooled from two or more independent experiments.