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bioRxiv · 10.1101/2025.07.16.665128

The EF-hand domain of MINDY3 is a Ubiquitin and RAD23 UBL-binding domain

Kulathu, Y.·Armstrong, L. A.·McFarland, M. R.·O'Dea, R.·Krutyholowa, R.·Gorka, M.·Carroll, T.·Glatt, S.
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Abstract

The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire length of the protein except for an atypical EF-hand insertion. We uncover this EF-hand (MINDY3EF-hand) to be a ubiquitin-binding domain with three distinct binding sites, enabling MINDY3 to bind and effectively cleave long polyubiquitin chains. Furthermore, the MINDY3EF-hand domain binds not only to polyubiquitin but also to the UBL domain of the proteasome shuttling and DNA repair factors RAD23A and RAD23B. The MINDY3EF-hand facilitates this interaction with RAD23s in cells and mediates MINDY3 recruitment to DNA damage sites, establishing this unique DUB as a potential regulator of cellular DNA damage responses. MINDY3 binds specifically to the UBL domain of RAD23s, and none of the other UBLs tested. The crystal structure of the MINDY3EF-hand:RAD23AUBL domain complex reveals the molecular basis for specificity. We find that MINDY3 can form a ternary complex with RAD23A/B and polyubiquitin, and our findings suggest a model wherein MINDY3 can deubiquitylate RAD23A/B-bound clients.

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Kulathu, Y., Armstrong, L. A., McFarland, M. R., O'Dea, R., Krutyholowa, R., Gorka, M., Carroll, T., Glatt, S.. 2025-07-21. The EF-hand domain of MINDY3 is a Ubiquitin and RAD23 UBL-binding domain. https://doi.org/10.1101/2025.07.16.665128

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