Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity

Maurer, Sigrun K.; Mayer, Matthias P.; Ward, Stephanie J.; Boudjema, Sana; Halawa, Mohamed; Zhang, Jiatong; Caulton, Simon G.; Emsley, Jonas; Dreveny, Ingrid

Abstract

Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1′ binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate “gatekeeper” residue in the S1′ site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Citation

Maurer, S. K., Mayer, M. P., Ward, S. J., Boudjema, S., Halawa, M., Zhang, J., Caulton, S. G., Emsley, J., & Dreveny, I. (2023). Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity. Journal of Biological Chemistry, 299(11), Article 105300. https://doi.org/10.1016/j.jbc.2023.105300

• Journal Article Type: Article
• Acceptance Date: Sep 19, 2023
• Online Publication Date: Sep 28, 2023
• Publication Date: 2023-11
• Deposit Date: Mar 26, 2025
• Publicly Available Date: Mar 26, 2025
• Journal: Journal of Biological Chemistry
• Electronic ISSN: 0021-9258
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 299
• Issue: 11
• Article Number: 105300
• DOI: https://doi.org/10.1016/j.jbc.2023.105300
• Keywords: ubiquitin-specific protease, crystal structure, cysteine protease, deubiquitylation (deubiquitination), protease, selectivity, ubiquitin, deubiquitinase, fusion tag,
• Public URL: https://nottingham-repository.worktribe.com/output/25644502
• Publisher URL: https://www.jbc.org/article/S0021-9258(23)02328-1/fulltext
• Additional Information: This article is maintained by: Elsevier; Article Title: Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity; Journal Title: Journal of Biological Chemistry; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.jbc.2023.105300; Content Type: article; Copyright: © 2023 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.