Structural insights into the interaction between testis‐specific Y‐encoded‐like protein 5 and ubiquitin‐specific protease 7

Abstract The Alternative Lengthening of Telomeres (ALT) mechanism enables telomere maintenance, contributing to the immortality of certain cancer cells. Disrupting the interaction between testis‐specific Y‐encoded‐like protein 5 (TSPYL5) and ubiquitin‐specific protease 7 (USP7) has emerged as a promising strategy to target ALT‐dependent cancers. While the N‐terminal MATH domain of USP7 mediates the protein interaction, the regions of TSPYL5 involved in binding remain unclear. Here, we present a structural analysis of the TSPYL5–USP7 interaction to guide targeted therapeutic strategies. We showed that TSPYL5 is intrinsically disordered, with an unfolded N‐terminal region and partial structure in the C‐terminal half. In vitro , recombinantly expressed TSPYL5 binds USP7 with nanomolar affinity and is prone to C‐terminal truncation. However, the truncated form retained a similar binding affinity for USP7, suggesting the primary interaction site resides in the N‐terminal region of TSPYL5. We identified three key binding hotspots within TSPYL5: residues 65–97, residues 210–262, and residues 368–388. Moreover, TSPYL5 forms trimers that further assemble into hexamers. This study provides the first structural and quantitative analysis of the TSPYL5–USP7 interaction, highlighting these three binding sites. These findings lay the groundwork for the development of novel inhibitors targeting ALT‐dependent cancers.