Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase.
Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase.
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DOI：
10.1074/jbc.m112.353748
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发表时间：
2012-05-18
期刊：
影响因子：
--
通讯作者：
Shaw GS
中科院分区：
文献类型：
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作者：
Spratt DE;Wu K;Kovacev J;Pan ZQ;Shaw GS
背景：Rbx1/ROC1是一种E3连接酶适配蛋白，与E2酶CDC34一起发挥作用。结果：核磁共振和生化数据表明，Rbx1/ROC1与CDC34∼泛素的结合强度是CDC34的50倍。结论：Rbx1/roc1细胞选择性地募集E2∼泛素，并在泛素转移后释放E2。意义：直接证据显示E3连接酶优先识别E2∼泛素复合体。环E3连接酶是一种蛋白质，它必须选择性地招募一个E2结合酶并促进泛素转移到底物上。目前尚不清楚E3环连接酶如何区分裸露的E2酶和E2∼泛素结合形式，也不清楚这种区别在泛素转移时是如何改变的。环盒蛋白1(Rbx1/ROc1)是在Skp1/cullin-1/F-box(SCF)E3泛素连接酶复合体中发现的一种关键蛋白，与E2泛素结合酶CDC34一起发挥作用。Rbx1/ROC1的溶液结构显示一个由三个结构锌离子(均方根偏差0.30±0.04Å)稳定的球形环状结构域(残基40-108)和一个无序的N末端(残基12-39)。滴定数据显示，Rbx1/ROC1优先招募泛素结合形式的CDC34，并且这种相互作用是未结合CDC34的50倍。此外，核磁共振和生化分析证实了Rbx1/ROC1螺旋α2上的残基是结合和激活CDC34CDC34∼泛素化所必需的。综上所述，这项工作提供了第一个直接的结构和生化证据，表明E3环连接酶Rbx1/ROc1的多泛素化需要优先招募E2∼泛素复合体，然后在泛素转移到底物或泛素链时释放未结合的E2蛋白。
Background: Rbx1/ROC1 is an E3 ligase adaptor protein that functions with the E2 enzyme CDC34. Results: NMR and biochemical data show that Rbx1/ROC1 binds CDC34∼ubiquitin 50-fold tighter than CDC34. Conclusion: Rbx1/ROC1 selectively recruits E2∼ubiquitin and releases the E2 after ubiquitin transfer. Significance: Direct evidence is shown for preferential recognition of an E2∼ubiquitin complex by an E3 ligase. RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.