Mechanism of USP21 autoinhibition and histone H2AK119 deubiquitination.

Monoubiquitinated histone H2A lysine 119 (H2AK119ub) is a modification associated with transcriptional silencing and heterochromatin formation. Ubiquitin-specific protease 21 (USP21), one of four major H2AK119-specific deubiquitinating enzymes (DUBs), plays critical roles in diverse cellular processes. However, the mechanisms by which USP21 specifically deubiquitinates H2AK119ub and is regulated are unknown. We determined the cryo-EM structure of the USP21 catalytic domain bound to an H2AK119ub nucleosome, which revealed a recognition mode that differs from that of other H2AK119-specific DUBs. We unexpectedly found that the N-terminal IDR of USP21 inhibits the enzyme's activity. Using AlphaFold-Multimer to perform a virtual screen of USP21 interactors, we identified kinases that phosphorylate the USP21 IDR and thereby relieve autoinhibition. AlphaFold3 modeling of USP21 suggests a structural model for autoinhibition. AlphaFold analysis suggests that phosphorylation-regulated autoinhibition may be a feature of various USP enzymes. These findings shed light on the mechanisms of H2AK119 deubiquitination and reveal a previously unexplored mode of phosphorylation-dependent DUB autoregulation.