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

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNF168 is an E3 ubiquitin ligase essential for the DNA damage response.
• RNF168 recruitment to double-strand breaks (DSBs) depends on ubiquitylated targets via its UDM1 and UDM2 regions.

Purpose of the Study:

• To elucidate the structural basis of RNF168's interaction with Lys63-linked ubiquitin chains.
• To determine the distinct roles of UDM1 and UDM2 in RNF168 recruitment to DSBs.

Main Methods:

• X-ray crystallography to determine the structures of UDM1-diUb and UDM2ΔC-diUb complexes.
• Biochemical assays to analyze ubiquitin-binding mechanisms.
• Site-directed mutagenesis in U2OS cells to assess RNF168 recruitment to DSBs.

Main Results:

• Crystal structures reveal UDM1 and UDM2ΔC as α-helices binding to Lys63-linked diubiquitin (K63-Ub2).
• Specific binding to distal and proximal ubiquitin moieties confers specificity for Lys63-linked chains.
• Mutations in UDM2, but not UDM1, significantly impair RNF168 accumulation at DSB sites.

Conclusions:

• RNF168 utilizes distinct ubiquitin-binding modules (UDM1 and UDM2) for target recognition.
• UDM2 plays a primary role in recruiting RNF168 to DNA damage sites through interactions with ubiquitylated targets.