UBCH5B~UBIQUITIN-HECTNEDD4L COMPLEX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 了解泛素(Ub)结合的一个关键问题是Ub是如何在E1-E2-E3级联中的酶之间转移的。对于Hect(与E6AP C-末端同源)类的E3，~40 kDa的C-末端Hect结构域与反应性的硫酯连接的E2~Ub结合(这里的“~”是指硫酯或类硫酯的共价键)。然后发生跨硫基化反应，Ub从E2催化半胱氨酸转移到Hect结构域催化半胱氨酸。因此，对于E2到E3的Ub转移来说，Ub的C末端和E2和Hect结构域的活性位点必须并列。 在人类中，近30个Hect E3通过与不同的E2选择性相互作用而带电，随后催化靶标泛素化。例如，Hect E3 NEDD4L已经被证明结合和接收来自包括UbcH5B和Ube2E3的E2的子集的Ub。NEDD4L的一个公认的下游功能是通过上皮钠通道(ENaC)的泛素化来调节血压。 尽管NEDD4L和其他Hect E3具有重要的生理作用，但其基本的酶机制仍然知之甚少。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结构域有两个结构“叶”，由一个灵活的连接子连接。N-末端的“N-叶”与E2催化半胱氨酸远端的部分E2结合。C-端“C-叶”含有Hect催化半胱氨酸，它从E2处接收Ub，形成硫酯连接的E3~Ub络合物。在包含UbcH7和E6AP的Hect结构域的E2-Hect结构域复合体的唯一晶体结构中，一个41？的间隙分隔了E2和E3半胱氨酸。因此，我们正在研究E_2~泛素络合物中Hect E_3络合物的结构，以期了解泛素转移的基本机制。

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.