Sims Joshua J, Haririnia Aydin, Dickinson Bryan C, Fushman David, Cohen Robert E
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
Nat Struct Mol Biol. 2009 Aug;16(8):883-9. doi: 10.1038/nsmb.1637. Epub 2009 Jul 20.
Ubiquitin (denoted Ub) receptor proteins as a group must contain a diverse set of binding specificities to distinguish the many forms of polyubiquitin (polyUb) signals. Previous studies suggested that the large class of ubiquitin-associated (UBA) domains contains members with intrinsic specificity for Lys63-linked polyUb or Lys48-linked polyUb, thus explaining how UBA-containing proteins can mediate diverse signaling events. Here we show that previously observed Lys63-polyUb selectivity in UBA domains is the result of an artifact in which the dimeric fusion partner, glutathione S-transferase (GST), positions two UBAs for higher affinity, avid interactions with Lys63-polyUb, but not with Lys48-polyUb. Freed from GST, these UBAs are either nonselective or prefer Lys48-polyUb. Accordingly, NMR experiments reveal no Lys63-polyUb-specific binding epitopes for these UBAs. We reexamine previous conclusions based on GST-UBAs and present an alternative model for how UBAs achieve a diverse range of linkage specificities.
泛素（标记为Ub）受体蛋白作为一个群体，必须包含多种不同的结合特异性，以区分多种形式的多聚泛素（polyUb）信号。先前的研究表明，一大类泛素相关（UBA）结构域包含对赖氨酸63连接的多聚泛素或赖氨酸48连接的多聚泛素有内在特异性的成员，从而解释了含UBA的蛋白质如何介导多种信号事件。在这里，我们表明，先前在UBA结构域中观察到的赖氨酸63-多聚泛素选择性是一种假象的结果，其中二聚体融合伙伴谷胱甘肽S-转移酶（GST）将两个UBA定位为与赖氨酸63-多聚泛素有更高亲和力、更强烈的相互作用，但与赖氨酸48-多聚泛素没有这种相互作用。从GST中释放出来后，这些UBA要么是非选择性的，要么更喜欢赖氨酸48-多聚泛素。因此，核磁共振实验没有揭示这些UBA的赖氨酸63-多聚泛素特异性结合表位。我们重新审视了基于GST-UBA的先前结论，并提出了一个关于UBA如何实现多种连接特异性的替代模型。
