Purpose: To investigate the structural features of wild-type and phospho-mimicking mutated XRCC4 protein, a protein involved in DNA double-strand break repair.
Materials and methods: XRCC4 with a HisTag were expressed by E. coli harboring plasmid DNA and purified. Phospho-mimicking mutants in which one phosphorylation site was replaced with aspartic acid were also prepared in order to reproduce the negative charge resulting from phosphorylation. The proteins were separated into dimers and multimers by gel filtration chromatography. Circular dichroism (CD) spectroscopy was performed in the region from ultraviolet to vacuum-ultraviolet. The CD spectra were analyzed with two analysis programs to evaluate the secondary structures of the wild-type and phospho-mimicked dimers and multimers.
Result and discussion: The proportion of β-strand in the wild-type dimers was very low, particularly in their C-terminal region, including the five phosphorylation sites. The secondary structure of the phospho-mimic hardly changed in the dimeric form. In contrast, the β-strand content increased and the α-helix content decreased upon multimerization of the wild-type protein. The structural change of multimers slightly depended on the phospho-mimic site. These results suggest that the β-strand structure stabilizes the multimerization of XRCC4 and it is regulated by phosphorylation at the C-terminal site in living cells.
Conclusion: An increase in the β-strand content in XRCC4 is essential for stabilization of the multimeric form through C-terminal phosphorylation, allowing the formation of the large double-strand break repair machinery.
Keywords: Circular dichroism; X-ray repair cross-complementing protein 4 (XRCC4); non-homologous end joining; protein phosphorylation; structural change.