UBCH5B~UBIQUITIN-HECTNEDD4L COMPLEX

UBCH5B~泛素-HECTNEDD4L 复合物

• 批准号: 8361696
• 负责人: BRENDA A SCHULMAN
• 金额: $ 2.74万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361696
• 关键词: Active Sites; Binding; C-terminal; Catalytic Domain; Charge; Complex; Cysteine; Distal; Enzymes; Epithelial; Funding; Grant; Human; Link; Lobe; N-terminal; National Center for Research Resources; Physiological; Principal Investigator; Reaction; Research; Research Infrastructure; Resources; Role; Sodium Channel; Source; Structure; UBE2D2 gene; Ubiquitin; Ubiquitin C; Ubiquitination; United States National Institutes of Health; blood pressure regulation; cost; epithelial Na+ channel; flexibility; structural biology; thioester

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A key question in understanding ubiquitin (Ub) conjugation is how Ub is transferred between enzymes in E1-E2-E3 cascades. For E3s in the HECT (Homologous to E6AP C-Terminus) class, the ~40 kDa C-terminal HECT domain binds a reactive thioester-linked E2~Ub (here "~" refers to thioester or thioester-like covalent linkage). Then a transthiolation reaction ensues, whereby Ub is transferred from the E2 catalytic Cys to the HECT domain catalytic Cys. Thus, the Ub C-terminus and the active sites of the E2 and HECT domain must all be juxtaposed for E2-to-E3 Ub transfer. In humans, nearly 30 HECT E3s become charged by selective interactions with distinct E2s, and subsequently catalyze target ubiquitination. For example, the HECT E3 NEDD4L has been shown to bind and receive Ub from a subset of E2s including UbcH5B and Ube2E3. A well-recognized downstream function of NEDD4L is regulation of blood pressure through ubiquitination of the Epithelial Sodium Channel (ENaC). Despite important physiological roles of NEDD4L and other HECT E3s, their fundamental enzymatic mechanisms remain poorly understood. A particularly vexing question is how a HECT domain and a specific Ub-loaded E2 interact to promote Ub transfer. Prior studies showed that HECT domains have two structural "lobes" tethered by a flexible linker. The N-terminal "N-lobe" binds part of an E2 distal from the E2 catalytic Cys. The C-terminal "C-lobe" contains the HECT catalytic Cys, which receives Ub from the E2 to form a thioester-linked E3~Ub complex. In the only crystal structure of an E2-HECT domain complex, containing UbcH7 and the HECT domain of E6AP, a 41 ¿ gap separates the E2 and E3 cysteines. Thus, we are studying structures of HECT E3 complexes in complexes with E2~ubiquitin in order to understand fundamental mechanisms of ubiquitin transfer.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 理解泛素（Ub）结合的一个关键问题是Ub如何在E1-E2-E3级联中的酶之间转移。 对于HECT（同源物至E6 AP C末端）类别中的E3，~40 kDa C末端HECT结构域结合反应性硫酯连接的E2~Ub（此处“~”是指硫酯或硫酯样共价键）。 然后进行巯基转移反应，由此将Ub从E2催化Cys转移到HECT结构域催化Cys。 因此，Ub C-末端和E2和HECT结构域的活性位点必须全部并置，以用于E2至E3 Ub转移。 在人类中，近30个HECT E3通过与不同E2的选择性相互作用而带电，随后催化靶泛素化。 例如，HECT E3 NEDD 4L已显示结合并接收来自包括UbcH 5 B和Ube 2 E3的E2子集的Ub。NEDD 4L的一个公认的下游功能是通过上皮钠通道（ENaC）的泛素化调节血压。 尽管NEDD 4L和其他HECT E3具有重要的生理作用，但其基本酶促机制仍知之甚少。 一个特别令人烦恼的问题是HECT结构域和特定的Ub负载的E2如何相互作用以促进Ub转移。 先前的研究表明，HECT结构域具有由柔性接头栓系的两个结构“叶”。 N-末端“N-叶”结合E2催化Cys远端的E2的一部分。C-末端“C-半段”含有HECT催化Cys，其从E2接收Ub以形成硫酯连接的E3-Ub复合物。 在含有UbcH 7和E6 AP的HECT结构域的E2-HECT结构域复合物的唯一晶体结构中，41 <$的间隙将E2和E3半胱氨酸分开。 因此，我们正在研究与E2-泛素复合的HECT E3复合物的结构，以了解泛素转移的基本机制。