Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination

Zhang, T.; Zheng, J.; Tong, Z.; Deng, Z.; He, Z.; Wu, X.; Wang, M.; Du, Y.; Xu, Z.; Tao, S.; Tian, X.; Deng, H.; Pan, M.; Ai, H.; Liu, L.

doi:10.1101/2025.04.25.650627

bioRxiv · 10.1101/2025.04.25.650627

Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination

Zhang, T.·Zheng, J.·Tong, Z.·Deng, Z.·He, Z.·Wu, X.·Wang, M.·Du, Y.·Xu, Z.·Tao, S.·Tian, X.·Deng, H.·Pan, M.·Ai, H.·Liu, L.

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Abstract

Protein ubiquitination plays a critical role in regulating fundamental biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for its role in targeting proteins for degradation, ubiquitination can also modulate enzymatic activity. However, all of the known mechanisms of such modulation involve spatially constrained mechanisms that take place near the enzyme-substrate interfaces. Here, we report an unprecedented regulatory paradigm mediated by ubiquitination that is located distal to the enzyme-substrate interface but can still activate the polycomb repressive deubiquitinase (PR-DUB) complex. Using cryo-electron microscopy, molecular dynamics (MD) simulations, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and kinetics assays, we determined that the ASXL1 K351 monoubiquitination promotes PR-DUB-mediated nucleosomal H2AK119Ub deubiquitination by stabilizing the catalytic pocket of PR-DUB, causing significant increase in maximal catalytic velocity (Vmax) but almost no change in the substrate binding affinity (Km). Structural analysis revealed that the ubiquitin moiety at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, acting as an intramolecular "glue" that constrains the conformational dynamics of these subunits without altering the nucleosome binding interface. Further MD and HDX-MS results demonstrated the monoubiquitination of ASXL1 K351 enables PR-DUB to effectively maintain a spatial arrangement of the catalytic conformation that closely aligns with substrate cleavage. This study expands our understanding of the mechanisms of ubiquitin function to include intramolecular fastening with a molecular gluing effect and provides a deeper mechanistic understanding of PR-DUB activation in nucleosomal H2AK119Ub removal.

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Zhang, T., Zheng, J., Tong, Z., Deng, Z., He, Z., Wu, X., Wang, M., Du, Y., Xu, Z., Tao, S., Tian, X., Deng, H., Pan, M., Ai, H., Liu, L.. 2025-04-27. Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination. https://doi.org/10.1101/2025.04.25.650627

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