Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11’s Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation.

聚泛素链将蛋白质底物引导至26S蛋白酶体，在那里它们在ATP依赖的底物降解过程中被去泛素酶Rpn11去除。快速脱泛素是有效降解所必需的，但必须限于与ATPase马达接合的固定底物，以防止过早泛素链去除和底物逸出。在这里，我们揭示了酿酒酵母Rpn11的泛素结合结构。以及底物降解和去泛素化的机械化学耦合机理。泛素结合诱导Rpn11的Insert-1环从非活动的闭合状态向活动的β发夹结构转变。此开关限制了去泛素化速率，并通过机械底物易位到AAA +电机中而大大加速。Rpn11的去泛素作用和ATPase的泛素作用处于直接竞争中。因此，Rpn11的AAA +电机驱动加速对于确保仅从固定的底物上去除聚泛素链并确保其足够快的速度以防止其共同降解至关重要。