Poster Presentation Inaugural Australian Ubiquitin Summit 2025

ZnF-UBP domains as modulators of USP activity and ubiquitin turnover  (#136)

Jack JA Alexandrovics 1 , Rashmi RA Agrata 1 , Philipp Schenk 1 , Anthony Cerra 1 , Ueli Nachbur 1 , Jeff Babon 1 , David Komander 1
  1. Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

The ZnF-UBP domain is a ubiquitin-binding domain (UBD) found in 13 enzymes, including 11 USP family deubiquitinases. The ZnF-UBP domain has been described to bind the unconjugated C-terminus of free ubiquitin [1], a unique feature among UBDs that suggests a distinctive regulatory mechanism. However, limited further characterisation has shown that the domains vary in function across the family [2]. Notably, seven ZnF-UBP-containing USPs are reported to act on ubiquitinated histones [3], hinting at an interesting link between ubiquitin dynamics, proteostasis, and gene regulation [4, 5]. The overall role of the ubiquitin-binding ZnF-UBP domain in regulating enzymatic activity, as well as its direct link to histone substrates, remains unclear [6]. 

We characterised all human ZnF-UBP domains for their ability to bind ubiquitin, with crystal structures of the USP16 and USP49 ZnF-UBP domains revealing distinct ubiquitin recognition capacities. Among ZnF-UBP-containing nucleosome DUBs, we observed variable effects on nucleosome binding, suggesting that shared structural motifs may mediate nucleosome interaction in both USP49 and USP16. Using USP16 mutants and engineered ubiquitin variants, we identified a coordinated “in concert” mechanism linking the ZnF-UBP and catalytic domains of USP16—each capable of acting independently, but requiring interplay for full catalytic efficiency. This uncovered a novel role for the ZnF-UBP domain in promoting product release by facilitating the clearance of cleaved ubiquitin from the active site. Finally, we extended this analysis across the USP family, revealing a finely tuned kinetic balance between the ZnF-UBP domain and the USP domain that underpins enzymatic regulation.

  1. [1] Reyes-Turcu, F. E., Horton, J. R., Mullally, J. E., Heroux, A., Cheng, X. & Wilkinson, K. D. (2006). The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin. Cell, 124(6), 1197–1208. https://doi.org/10.1016/j.cell.2006.02.038
  2. [2] Agafonov, D. E., Kastner, B., Dybkov, O., Hofele, R. V., Liu, W.-T., Urlaub, H., Lührmann, R. & Stark, H. (2016). Molecular architecture of the human U4/U6.U5 tri-snRNP. Science, 351(6280), 1416–1420. https://doi.org/10.1126/science.aad2085
  3. [3] Mattiroli, F. & Penengo, L. (2021). Histone Ubiquitination: An Integrative Signaling Platform in Genome Stability. Trends in Genetics. https://doi.org/10.1016/j.tig.2020.12.005
  4. [4] Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A., Sowa, M. E., Rad, R., Rush, J., Comb, M. J., Harper, J. W. & Gygi, S. P. (2011). Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome. Molecular Cell, 44(2), 325–340. https://doi.org/10.1016/j.molcel.2011.08.025
  5. [5] Hanna, J., Meides, A., Zhang, D. P. & Finley, D. (2007). A Ubiquitin Stress Response Induces Altered Proteasome Composition. Cell, 129(4), 747–759. https://doi.org/10.1016/j.cell.2007.03.042
  6. [6] Ai, H., He, Z., Deng, Z., Chu, G.-C., Shi, Q., Tong, Z., Li, J.-B., Pan, M. & Liu, L. (2024). Structural and mechanistic basis for nucleosomal H2AK119 deubiquitination by single-subunit deubiquitinase USP16. Nature Structural & Molecular Biology, 1–11. https://doi.org/10.1038/s41594-024-01342-2