Conserved disordered regions and ubiquitin-binding motifs within HUWE1 contribute to substrate targeting plasticity

E3 ligases confer substrate specificity within ubiquitin-dependent pathways. However, the pleiotropic HECT-type E3 ligase HUWE1 targets a biochemically and structurally diverse set of substrates through mechanisms that remain largely unknown. We develop a combinatorial cell-based system integrating global proteomics, proximity labeling, and reporter assays to systematically interrogate HUWE1 sequence features that enable substrate targeting. We define a robust HUWE1 activity signature consisting of putative substrates that accumulate upon HUWE1 impairment across multiple cell lines. By re-expressing a panel of HUWE1 deletion mutants, we find that both the largest intrinsically disordered region and the conserved ubiquitin-binding motifs contribute to substrate targeting. Structural modeling and domain analysis further suggest that HUWE1 achieves targeting plasticity through multivalent interactions, engaging both disordered and structured regions within its substrates. Together, our findings establish a framework for understanding how large, modular E3 ligases exploit intrinsic disorder and multivalency to shape their substrate repertoires.