Takahashi Tomio S, Hirade Yoshihiro, Toma Aya, Sato Yusuke, Yamagata Atsushi, Goto-Ito Sakurako, Tomita Akiko, Nakada Shinichiro, Fukai Shuya
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan.
Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo, 113-0032, Japan.
Nat Commun. 2018 Jan 12;9(1):170. doi: 10.1038/s41467-017-02345-y.
The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). The recruitment of RNF168 by ubiquitylated targets involves two distinct regions, Ub-dependent DSB recruitment module (UDM) 1 and UDM2. Here we report the crystal structures of the complex between UDM1 and Lys63-linked diUb (K63-Ub) and that between the C-terminally truncated UDM2 (UDM2ΔC) and K63-Ub. In both structures, UDM1 and UDM2ΔC fold as a single α-helix. Their simultaneous bindings to the distal and proximal Ub moieties provide specificity for Lys63-linked Ub chains. Structural and biochemical analyses of UDM1 elucidate an Ub-binding mechanism between UDM1 and polyubiquitylated targets. Mutations of Ub-interacting residues in UDM2 prevent the accumulation of RNF168 to DSB sites in U2OS cells, whereas those in UDM1 have little effect, suggesting that the interaction of UDM2 with ubiquitylated and polyubiquitylated targets mainly contributes to the RNF168 recruitment.
E3泛素（Ub）连接酶RNF168在对双链断裂（DSB）的DNA损伤反应起始过程中发挥关键作用。RNF168被泛素化靶标招募涉及两个不同区域，即Ub依赖性DSB招募模块（UDM）1和UDM2。在此，我们报道了UDM1与赖氨酸63连接的双泛素（K63-Ub）复合物以及C端截短的UDM2（UDM2ΔC）与K63-Ub复合物的晶体结构。在这两种结构中，UDM1和UDM2ΔC均折叠成单一α螺旋。它们与远端和近端Ub部分的同时结合为赖氨酸63连接的Ub链提供了特异性。对UDM1的结构和生化分析阐明了UDM1与多泛素化靶标之间的Ub结合机制。UDM2中Ub相互作用残基的突变可阻止RNF168在U2OS细胞中积累至DSB位点，而UDM1中的突变影响很小，这表明UDM2与泛素化和多泛素化靶标的相互作用主要有助于RNF168的招募。
