Durfee Larissa A, Kelley Melissa L, Huibregtse Jon M
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA.
J Biol Chem. 2008 Aug 29;283(35):23895-902. doi: 10.1074/jbc.M804069200. Epub 2008 Jun 26.
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 alpha1-helix and the beta1-beta2 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.
E1酶和E2酶协同催化泛素（Ub）及泛素样蛋白（Ubls）缀合反应的起始步骤。ISG15是一种由α/β干扰素诱导产生的Ubl，负责ISG15缀合反应的E1酶和E2酶分别是Ube1L和UbcH8。UbcH7是与UbcH8亲缘关系最近的E2酶，但它在体内的ISG15缀合反应中不起作用，而据报道UbcH7和UbcH8在Ub缀合反应中均发挥作用。对野生型和嵌合型E2酶进行动力学分析，以确定Ube1L优先激活UbcH8的依据，并确定UbcH8是否能被Ube1L和E1（Ub）（Ube1）同等程度地激活。米氏常数（K(m)）测定证实，Ube1L对UbcH8的偏好远高于UbcH7（K(m)相差29倍），这与E1（Ub）对UbcH7的偏好高于UbcH8的情况类似（K(m)相差36倍）。对嵌合型E2酶的硫酯分析确定了UbcH8第1至39位残基内的两个结构元件，它们在决定Ube1L-UbcH8特异性方面起主要作用：α1螺旋和β1-β2区域。Ube1L的C末端泛素折叠结构域（UFD）是将ISG15转移至UbcH8以及Ube1L与UbcH8结合所必需的。用E1（Ub）的UFD替换Ube1L的UFD会导致ISG15优先转移至UbcH7。这些结果共同表明，Ube1L基于Ube1L的UFD与UbcH8 N末端区域内决定因素之间的特定相互作用，区分UbcH8和与之亲缘关系密切的Ub E2酶。
