The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates in vitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1 UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.

E3 连接酶 E6AP/UBE3A 在底物受体 hRpn10/S5a/PSMD4 的 RAZUL 结构域提供的 26S 蛋白酶体中具有专用结合位点。在 RAZUL 序列相似性的指导下，我们在这里测试并证明 E6AP AZUL 与蛋白酶体穿梭因子 UBQLN1/2 的 UBA 瞬时结合。尽管结合亲和力较弱，但 E6AP AZUL在体外被招募到 UBQLN2 生物分子凝聚物中，并且 E6AP在纤维素中与 UBQLN1/2 相互作用. 稳态和转移核 Overhauser 效应 (NOE) 实验表明 AZUL 与 UBQLN1 UBA 直接相互作用。将通过 NOE 光谱 (NOESY) 数据识别的分子间接触与 AlphaFold2-Multimer 预测相结合，以产生 AZUL:UBA 模型结构。我们还确定了一个与 UBQLN1/2 UBA 直接相邻的寡聚化结构域（UBA 相邻 [UBAA]），它是 α-螺旋的，并且通过 AZUL 与 UBA 的结合进行变构重构。这些数据导致通过 AZUL:UBA 相互作用将 E6AP 募集到 UBQLN1/2 的模型，并提供有关凝聚物和细胞中相互作用的结合要求的基本信息。