K27-Linked Diubiquitin Inhibits UCHL3 via an Unusual Kinetic Trap

Functional analysis of lysine 27-linked ubiquitin chains (^K27Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to ^K27Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic ^K27Ub₂, we unexpectedly discover that free ^K27Ub₂ and ^K27Ub₂-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that ^K27Ub₂ can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and ^K27Ub chains likely sense and regulate each other in cells.