Structural insights into two distinct binding modules for Lys63-linked polyubiquitin chains in RNF168

Tomio S Takahashi,Atsushi Yamagata,Yusuke Sato,Shinichiro Nakada,Yoshihiro Hirade,Sakurako Goto-Ito,Shuya Fukai,Aya Toma,Akiko Tomita

doi:10.1038/s41467-017-02345-y

Tomio S Takahashi, Atsushi Yamagata + Show 7 more

Open Access

https://doi.org/10.1038/s41467-017-02345-y

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Abstract

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-Ub2) and that between the C-terminally truncated UDM2 (UDM2ΔC) and K63-Ub2. 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.

Highlights

The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs)
UMI and MIU1 are included in a module called UDM (Ub-dependent DSB recruitment module) 1, which is located in the N-terminal part of RNF168
Previous studies have shown that RNF168 UDM1 comprises UMI23 and MIU114–16 as Ub-binding motifs

Summary

Introduction

The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). The E3 Ub ligase RNF168 recognizes these polyubiquitylated proteins and ubiquitylates histone H2A, which serves as a recruitment signal for 53BP114–19. Recognition of ubiquitylated products by RNF168 relies on three distinct UBDs: MIU (motif interacting with Ub) 1, MIU2 and UMI (Ubinteracting motif [UIM]- and MIU-related UBD) (Fig. 1). These domains are essential for recruitment of RNF16814–16, 23. UMI and MIU1 are included in a module called UDM (Ub-dependent DSB recruitment module) 1, which is located in the N-terminal part of RNF168.

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