Structural study of wild-type and phospho-mimic XRCC4 dimer and multimer proteins using circular dichroism spectroscopy

Abstract

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
• structural change
• non-homologous end joining
• protein phosphorylation
• X-ray repair cross-complementing protein 4 (XRCC4)

Acknowledgements

The authors thank Dr. S. Fujiwara and Dr. T. Matsuo at QST for their helpful support in sample preparation. The CD-spectroscopy experiments were performed with approval of the Hiroshima Synchrotron Radiation Center of Hiroshima University (proposal numbers: 17AG033, 17BG025, 18AG008, 18BG018, 19AG033, 19BG021).

Author contributions

K. N. and M. H. performed all experiments and analyzed CD data; Y. I. supported CD analysis; Y. I., K. F. and K. M. supported CD experiments in HiSOR. Y. M. provided XRCC4 expression plasmids and fundings of the project, A. Y. conceived the project and supervised all aspects of the project; and all authors contributed to manuscript editing.

Disclosure statement

No potential conflict of interest was reported by the author(s). The authors alone are responsible for the content and writing of this paper.

Notes

1 Lys298 in the 334 amino acid form is replaced by Asn298-Ser299-Arg300 in the 336 amino acid form. We used the 336 amino acid form and, therefore, refer to amino acids numbered according to the 336 amino acid form.

Additional information

Funding

This work was supported by JSPS KAKENHI Grant Number JP15H02817.

Notes on contributors

Kai Nishikubo

Kai Nishikubo is a Ph.D. student at Ibaraki University in Mito, Ibaraki, Japan. He is working on a project of structural studies of DNA repair proteins using synchrotron radiation techniques.

Maho Hasegawa

Maho Hasegawa is a master’s student at Ibaraki University. Her major is biochemistry and structural analyses of proteins.

Yudai Izumi

Yudai Izumi, Ph.D., is a principal investigator at Institute for Quantum Life Science (iQLS), QST in Tokai, Ibaraki, Japan. His experience and expertise in spectroscopies for biological applications stand out in the synchrotron radiation research field.

Kentaro Fujii

Kentaro Fujii, Ph.D., is a senior scientist at iQLS, QST, and has dedicated his expertise on surface science for the construction of X-ray beamlines in a new synchrotron radiation facility in Sendai, Japan.

Koichi Matsuo

Koichi Matsuo, Ph.D., is an associate professor at Hiroshima Synchrotron Radiation Center in Hiroshima University, Higashi-Hiroshima, Japan. He has superior performance of VUV-CD spectroscopy and leads many researchers for successful analyses of biomolecular structures.

Yoshihisa Matsumoto

Yoshihisa Matsumoto, Ph.D., is an associate professor at the Laboratory for Zero-Carbon Energy, Institute of Innovative Research, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan. He is an authority of DNA repair proteins and has great achievements on DNA end-joining proteins such as DNA-PK or XRCC4-ligase IV complex.

Akinari Yokoya

Akinari Yokoya, Ph.D., is a principal investigator of Quantum and System Biology Group at iQLS, QST in Tokai. He is also a professor at Ibaraki University under the cross-appointment system to educate undergraduate and graduate students about radiation biophysics.