The E3 ligase HUWE1 interacts with ubiquitin non‐covalently via key residues in the HECT domain.

HUWE1, a HECT E3 ligase, is critical for processes like protein degradation and tumor development. Contrary to previous findings which suggested minimal non‐covalent interactions between the HUWE1 HECT domain and ubiquitin, we identified a non‐covalent interaction between the HUWE1 HECT N‐lobe and ubiquitin using NMR spectroscopy, revealing a conserved ubiquitin‐binding mode shared across HECT E3 ligases. Molecular dynamics simulations not only confirmed the stability of this interaction but also uncovered conformational changes in key residues, which likely influence binding affinity. Additionally, we highlighted the roles of both conserved and unique residues in ubiquitin binding. These findings advance our understanding of the interactions between the HUWE1 HECT domain and ubiquitin, and highlight potential targets for therapeutic intervention in the ubiquitin‐proteasome pathway. [ABSTRACT FROM AUTHOR]