9I6A

Crystal structure of human Cdc20 bound to synthetic D-box peptide D7

9I6A の概要

• エントリーDOI: 10.2210/pdb9i6a/pdb
• 分子名称: Cell division cycle protein 20 homolog, ALA-PRO-0JY-GLY (3 entities in total)
• 機能のキーワード: apc/c, degron, d-box, ligase
• 由来する生物種: Homo sapiens (human); 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 55753.57

構造登録者

Eapen, R.,Okoye, C.,Stubbs, C.,Schimpl, M.,Tischer, T.,Fisher, E.J.,Zacharopoulou, M.,Ferrer, F.,Barford, D.,Spring, D.,Lindon, C.,Phillips, C.,Itzhaki, L.S. (登録日: 2025-01-29, 公開日: 2025-02-12, 最終更新日: 2025-09-17)

主引用文献

Eapen, R.,Okoye, C.,Stubbs, C.,Schimpl, M.,Tischer, T.,Fisher, E.J.,Zacharopoulou, M.,Ferrer, F.,Barford, D.,Spring, D.R.,Lindon, C.,Phillips, C.,Itzhaki, L.S.
Development of D-box peptides to inhibit the anaphase-promoting complex/cyclosome.
Elife, 14:-, 2025

PubMed Abstract: E3 ubiquitin ligases engage their substrates via 'degrons' - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are difficult to inhibit via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase-promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the 'destruction box' (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket that contributes to the D-box binding site on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20-peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/C and are more potent than the small-molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially for harnessing the APC/C for targeted protein degradation.

PubMed: 40888475
DOI: 10.7554/eLife.104238

実験手法

X-RAY DIFFRACTION (1.915 Å)