Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling
Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling
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DOI：
10.1074/jbc.m309184200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Fushman, D
中科院分区：
文献类型：
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作者：
Varadan, R;Assfalg, M;Fushman, D
通过共价连接一个或一系列泛素分子，对底物蛋白进行翻译后修饰，从而调节多种细胞事件。 （多）泛素化的结果取决于参与多泛素链形成的特定赖氨酸残基。 Lys(48) 连接的链充当蛋白酶体降解的通用信号，而 Lys(63) 连接的链充当多个非降解过程中的特定信号。尽管预计交替连接的多聚泛素链之间的功能多样性依赖于链构象/拓扑的连接依赖性差异，但缺乏支持该模型的直接结构证据。在这里，我们使用 NMR 方法来确定 Lys(63) 连接的双泛素链的结构。该结构的特点是延伸构象，泛素单元上的疏水残基 Leu(8)、Ile(44) 和 Val(70) 之间没有直接接触。该结构与 Lys(48) 连接的双泛素观察到的闭合构象形成对比，其中这些残基形成域间界面（Cook, W. J.、Jeffrey, L. C.、Carson, M.、Zhijian, C. 和 Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471 ；Varadan, R., Walker, O., Pickart, C. 和 Fushman, D. (2002) J. Biol. 324, 637-647)。与 Lys(63) 连接的双泛素的开放构象一致，我们的结合研究表明，该链中的两个泛素结构域都可以独立地结合 HHR23A 的泛素相关结构域，并且以类似于单泛素的模式结合。相比之下，Lys(48) 连接的双泛素以不同的、更高亲和力的模式结合，该模式尚未确定。这是第一个实验证据表明交替连接的多聚泛素链采用不同的构象。
Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys(48)-linked chains act as a universal signal for proteasomal degradation, whereas Lys(63)-linked chains act as a specific signal in several nondegradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys(63)-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu(8), Ile(44), and Val(70) on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys(48)- linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys(48)- linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.