The basis for selective E1-E2 interactions in the ISG15 conjugation system
The basis for selective E1-E2 interactions in the ISG15 conjugation system
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DOI：
10.1074/jbc.m804069200
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发表时间：
2008-08-29
影响因子：
4.8
通讯作者：
Huibregtse, Jon M.
中科院分区：
文献类型：
--
作者：
Durfee, Larissa A.;Kelley, Melissa L.;Huibregtse, Jon M.
E1和E2酶协调泛素(Ub)和泛素样蛋白(UBL)结合的第一步。ISG15是干扰素-α/β诱导的Ub1，与ISG15结合的E1和E2酶分别为Ube1L和UbcH8。UbcH7是与UbcH8关系最密切的E2，但在体内不参与ISG15的结合，而UbcH7和UbcH8都被报道在Ub的结合中发挥作用。对野生型和嵌合型E2s进行动力学分析，以确定Ube1L优先激活UbcH8的基础，以及Ube1L和E1(Ub)(Ube1)是否同样能激活UbcH8。K-m测定证实了Ube1L对UbcH8的强烈偏好(K-m差是UbcH7的29倍)，类似于E1(Ub)对UbcH7的偏好(K-m差36倍)。对嵌合E2的硫酯分析发现，UbcH8残基1-39中有两个结构元件在定义Ube1L-UbcH8特异性中起主要作用：α1-螺旋和β1-β2区域。Ube1L的C末端泛素折叠结构域(UFD)是ISG15转移到UbcH8和Ube1L与UbcH8结合所必需的。将Ube1L Ufd替换为来自E1Ub的Ufd，导致ISG15优先转移到UbcH7。综上所述，这些结果表明，Ube1L根据Ube1L UFD与UbcH8 N-末端区域内的决定因素之间的特定相互作用区分UbcH8和密切相关的Ub E2。
E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K-m determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K-m difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36- fold K-m difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha 1-helix and the beta 1-beta 2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.