Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B
Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B
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DOI：
10.1074/jbc.m115.685867
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发表时间：
2015-12-18
影响因子：
4.8
通讯作者：
Weissman, Allan M.
中科院分区：
文献类型：
--
作者：
Li, Shengjian;Liang, Yu-He;Weissman, Allan M.
环蛋白是E3泛素连接酶中最大的一类。与大多数环不同，AO7(RNF25)以惊人的高亲和力结合E2泛素结合酶UbcH5B(UBE2D2)。通过共结晶，我们定义了AO7与UbcH5B结合的独特方式。AO7含有一个结构独特的UbcH5B结合区(U5BR)，它由11个氨基酸连接到其环域上，形成围绕E2的钳制。U5BR与UbcH5B的一个区域广泛相互作用，该区域既不同于活性部位，也不同于环相互作用区，称为E2的背面。一个明显的悖论是，AO7钳与UbcH5B的高亲和力结合依赖于U5BR，降低了泛素化的速度。我们确定这是阻断泛素与UbcH5B背面的刺激性、非共价结合的结果。有趣的是，当非共价的背面泛素结合不能发生时，AO7钳现在可以提高泛素化的速度。AO7钳与UbcH5B的高亲和力结合也允许先前描述的和功能上重要的环突变体在环-E2界面上的共结晶。我们表明，对功能有显著影响的突变只极小地影响AO7环和UbcH5B之间的分子间相互作用，通过环-E2界面建立了高度的激活复杂性。
RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.