Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3.
Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3.
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DOI：
10.7554/elife.00828
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发表时间：
2013-08-08
期刊：
影响因子：
7.7
通讯作者：
Schulman BA
中科院分区：
文献类型：
--
作者：
Kamadurai HB;Qiu Y;Deng A;Harrison JS;Macdonald C;Actis M;Rodrigues P;Miller DJ;Souphron J;Lewis SM;Kurinov I;Fujii N;Hammel M;Piper R;Kuhlman B;Schulman BA
HECT E3 酶的泛素化可调节多种过程，包括肿瘤抑制、转录、蛋白质运输和降解。 HECT E3 使用两步机制将泛素连接到目标蛋白。第一步是由催化 HECT 结构域和 E2∼泛素中间体之间的相互作用引导，促进瞬时的、硫酯键合的 HECT∼泛素中间体的形成。在这里，我们报告连接的第二步是由 HECT E3 及其共价连接的泛素建立的独特催化结构介导的。 E3∼泛素-底物中间体的化学捕获代理的结构揭示了泛素和双叶HECT结构域之间的三向相互作用，定向E3∼泛素硫酯键进行连接，并限制底物结合结构域的位置以优先考虑目标赖氨酸进行泛素化。这些数据可实现 E2-至-E3-至底物泛素转移级联的可视化，并显示驱动多个反应的 HECT 特异性泛素相互作用如何通过主要的 E3 构象变化来重新利用以促进连接。 DOI：http://dx.doi.org/10.7554/eLife.00828.001 泛素是一种小蛋白质，可以与细胞中的其他“目标”蛋白质共价连接以影响其行为。泛素可以作为单拷贝或多聚泛素链与其靶标连接，其中多个泛素分子首尾相连，链的一端连接到靶蛋白。涉及 E1、E2 和 E3 酶的多步级联将泛素添加到其靶标中。这些酶的运作方式就像接力赛中的赛跑者一样，泛素就像一根接力棒，从 E1 传递到 E2，再到 E3，到达目标。 E3 酶是一种连接酶，可催化泛素与其靶标之间形成新的化学键。人体细胞中有大约 600 种不同的 E3 酶，可调节多种靶蛋白。一类主要的 E3 酶，称为 HECT E3，以独特的两步机制将泛素附着到其靶标上：E2 酶将泛素与 HECT E3 共价连接，形成复合物，随后将泛素转移到其靶蛋白上。泛素通常被添加到目标蛋白上的特定氨基酸赖氨酸上，但 HECT E3 如何执行这种转移的细节尚不清楚。为了解决这个问题，Kamadurai 等人。研究 Rsp5（酵母中的一种 HECT E3 连接酶）如何将泛素附着到称为 Sna3 的靶蛋白上。所有 HECT E3 都有一个结构域（HECT 结构域），可催化泛素转移至其靶蛋白。该结构域由两个子结构组成：C 叶和 N 叶，C 叶可以从 E2 接收泛素，然后自身与泛素连接。以前认为这些叶采用不同的相对方向，将泛素递送到不同靶蛋白上的位点（包括单个靶蛋白上的多个赖氨酸）。出乎意料的是，Kamadurai 等人。发现为了将泛素转移到 Sna3，Rsp5 采用了离散的 HECT 结构域结构，该结构创建了一个活性位点，其中通常分开的 C 叶和 N 叶部分与泛素分子结合在一起。这种架构还提供了一种机制，根据底物赖氨酸对该活性位点的可及性来决定哪些底物赖氨酸可以被泛素化。 Rsp5 的相同区域将泛素转移到 Sna3 以外的靶标，这表明存在统一的机制——Kamadurai 等人。 show 在两种相关的人类 HECT E3 连接酶中是保守的，可能会将泛素转移到其所有目标。因此，这些研究代表着在理解主要一类 E3 酶如何调节其靶标功能方面迈出了重要一步。 DOI：http://dx.doi.org/10.7554/eLife.00828.002
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI: http://dx.doi.org/10.7554/eLife.00828.001 Ubiquitin is a small protein that can be covalently linked to other, ‘target’, proteins in a cell to influence their behavior. Ubiquitin can be linked to its targets either as single copies or as polyubiquitin chains in which several ubiquitin molecules are bound end-on-end to each other, with one end of the chain attached to the target protein. A multi-step cascade involving enzymes known as E1, E2, and E3 adds ubiquitin to its targets. These enzymes function in a manner like runners in a relay, with ubiquitin a baton that is passed from E1 to E2 to E3 to the target. The E3 enzyme is a ligase that catalyzes the formation of a new chemical bond between a ubiquitin and its target. There are approximately 600 different E3 enzymes in human cells that regulate a wide variety of target proteins. A major class of E3 enzymes, called HECT E3s, attaches ubiquitin to its targets in a unique two-step mechanism: the E2 enzymes covalently link a ubiquitin to a HECT E3 to form a complex that subsequently transfers the ubiquitin to its target protein. The ubiquitin is typically added to a particular amino acid, lysine, on the target protein, but the details of how HECT E3s execute this transfer are not well understood. To address this issue, Kamadurai et al. investigate how Rsp5, a HECT E3 ligase in yeast, attaches ubiquitin to a target protein called Sna3. All HECT E3s have a domain—the HECT domain—that catalyzes the transfer of ubiquitin to its target protein. This domain consists of two sub-structures: the C-lobe, which can receive ubiquitin from E2 and then itself become linked to ubiquitin, and the N-lobe. These lobes were previously thought to adopt various orientations relative to each other to deliver ubiquitin to sites on different target proteins (including to multiple lysines on a single target protein). Unexpectedly, Kamadurai et al. find that in order to transfer the ubiquitin to Sna3, Rsp5 adopts a discrete HECT domain architecture that creates an active site in which parts of the C-lobe and the N-lobe, which are normally separated, are brought together with a ubiquitin molecule. This architecture also provides a mechanism that dictates which substrate lysines can be ubiquitinated based on how accessible they are to this active site. The same regions of Rsp5 transfer ubiquitin to targets other than Sna3, suggesting that a uniform mechanism—which Kamadurai et al. show is conserved in two related human HECT E3 ligases—might transfer ubiquitin to all its targets. These studies therefore represent a significant step toward understanding how a major class of E3 enzymes modulates the functions of their targets. DOI: http://dx.doi.org/10.7554/eLife.00828.002