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.

赖氨酸 27 连接的泛素链 ( ^K27 Ub) 的功能分析很困难，因为无法通过酶促方法制造它们，并且缺乏模型工具和底物。在这里，我们生成了一系列泛素 (Ub) 工具来研究去泛素化酶 UCHL3与赖氨酸 63 连接链和单 Ub 相比如何响应^K27 Ub 链。从UCHL3和合成^K27 Ub ₂复合物的晶体结构中，我们意外地发现游离的^K27 Ub ₂和^K27 Ub ₂结合底物是 UCHL3 的天然抑制剂。使用我们的 Ub 工具来分析 UCHL3 的活性，我们生成了抑制机制的定量动力学模型，我们发现^K27 Ub ₂可以共价抑制 UCHL3，通过与其催化半胱氨酸结合，并通过变构方式将其催化环紧紧锁定到位。基于这种抑制机制，我们提出 UCHL3 和^K27 Ub 链可能在细胞中相互感知和调节。