The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Its diverse substrates unify as proteasome targets by ubiquitination. We used cryogenic electron microscopy (cryo-EM) to study how human 26S proteasome interacts with M1-linked hexaubiquitin (M1-Ub₆) unanchored to a substrate and E3 ubiquitin ligase E6AP/UBE3A. Proteasome structures are available with model substrates extending through the RP ATPase ring and substrate-conjugated K63-linked ubiquitin chains present at inhibited deubiquitinating enzyme hRpn11 and the nearby ATPase hRpt4/hRpt5 coiled coil. In this study, we find M1-Ub₆ at the hRpn11 site despite the absence of conjugated substrate, indicating that ubiquitin binding at this location does not require substrate interaction with the RP. Moreover, unanchored M1-Ub₆ binds to this hRpn11 site of the proteasome with the CP gating residues in both the closed and opened conformational states.

26S 蛋白酶体专门用于调节蛋白质降解，由动态调节颗粒 (RP) 形成，动态调节颗粒 (RP) 覆盖在空心圆柱形核心颗粒 (CP) 上，底物在其中进行蛋白水解。其不同的底物通过泛素化统一为蛋白酶体靶标。我们使用低温电子显微镜 (cryo-EM) 研究人类 26S 蛋白酶体如何与未锚定到底物的 M1 连接六泛素 (M1-Ub ₆ ) 和 E3 泛素连接酶 E6AP/UBE3A 相互作用。蛋白酶体结构具有延伸穿过 RP ATPase 环的模型底物和存在于受抑制的去泛素化酶 hRpn11 和附近 ATPase hRpt4/hRpt5 卷曲线圈处的底物缀合的 K63 连接的泛素链。在这项研究中，我们在 hRpn11 位点发现了 M1-Ub _6，尽管不存在缀合底物，这表明泛素在该位置的结合不需要底物与 RP 相互作用。此外，未锚定的 M1-Ub ₆与处于闭合和开放构象状态下的 CP 门控残基的蛋白酶体的 hRpn11 位点结合。