ZnF-UBP domains regulate deubiquitinase activity by relieving ubiquitin product inhibition

Alexandrovics, J. A.; Agrata, R.; Schenk, P.; Cerra, A.; Nachbur, U.; Babon, J. J.; Komander, D.

doi:10.1101/2025.09.28.679104

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bioRxiv · 10.1101/2025.09.28.679104

ZnF-UBP domains regulate deubiquitinase activity by relieving ubiquitin product inhibition

Alexandrovics, J. A.·Agrata, R.·Schenk, P.·Cerra, A.·Nachbur, U.·Babon, J. J.·Komander, D.

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Abstract

Most ubiquitin specific protease (USP) deubiquitinases (DUBs) combine non-selective catalytic domains with one or multiple exo-domains that contribute substrate specificity and localisation, but are generally poorly characterised. Zinc-Finger UBP (ZnF-UBP) domains exist in 12 USP DUBs, yet their function is unclear. We here comprehensively analyse human ZnF-UBP domains, and reveal that 8 of 14 bind ubiquitin (Ub), via an unattached Ub C-terminal GlyGly motif. We focus on USP16, a nucleosome DUB with activity for Ub and Ub-like modifiers, and show that whilte its ZnF-UBP domain can bind substrates, it is also a crucial contributor to enzyme kinetics. Slow Ub release from the catalytic domain after cleavage causes product inhibition, which is overcome in cis by ZnF-UBP-mediated product release. Interestingly, supplying a high affinity product-capturing ZnF-UBP domain in trans, activates USP16 and other USP enzymes. Our data shows the importance of product inhibition as a regulatory mechanism in DUBs, and exemplifies the unappreciated role of exo-domains in regulating DUB function beyond substrate binding.

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Alexandrovics, J. A., Agrata, R., Schenk, P., Cerra, A., Nachbur, U., Babon, J. J., Komander, D.. 2025-09-29. ZnF-UBP domains regulate deubiquitinase activity by relieving ubiquitin product inhibition. https://doi.org/10.1101/2025.09.28.679104

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