Non-catalytic UBL2 domain directs deubiquitinase USP11 toward K48-linked polyubiquitin chains

Ubiquitin-specific proteases (USPs), comprising the largest deubiquitinase family, are generally thought to have poor discrimination of ubiquitin (Ub) linkage types, but a number of USPs show preference toward certain linkages. USP11, a USP-family member implicated in cancer and neurodegeneration, carries an atypical catalytic domain which is split into two segments through the insertion of a UBL2 domain and an intrinsically disordered region (IDR). In addition, the chain-type selectivity of USP11 remains unclear based on the conflicting data from in vitro and in vivo studies. Here, we identify an important role of the UBL2-IDR in altering the ability of USP11 to cleave K29, K33, and K48 chains, with K48 chain showing the most significant effect. Using in vitro studies with Ub-tetramer and ubiquitinated proteins as well as cell-based analyses, we demonstrate that UBL2 domain endows USP11 with a selectivity towards the K48-linked Ub chains. Importantly, this function of UBL2 is not observed in its paralogs USP4 and USP15, which display broad activities towards most chain types. By leveraging AI-based virtual screening, we have identified selective USP11 inhibitors, including the FDA-approved drugs Fenoldopam and Olanzapine and their analogs, which act through a unique chemical scaffold and display significant efficacy both in vitro and in cells. Our findings not only uncover a previously unrecognized mechanism of linkage selectivity within the USP family but also provide a robust platform for the rational design of USP11-targeted therapeutics, underscoring the critical role of non-catalytic domains in deubiquitinase regulation and offering promising avenues for therapeutic intervention.