One of the tools that we use to elucidate the mechanism of action of a protein is to determine its structure by X-ray crystallography. To facilitate this process the structural domains of the protein are defined by partial proteolysis in order to generate a version of the protein that is suitable for structure determination. We have successfully applied this approach in determining the structures of the EBNA1 DNA binding domain (both on its own and bound to DNA), several domains of RPA (the human single-stranded DNA binding protein) and the N-terminal target recognition domain of USP7 in complex with EBNA1, p53 and Mdm2 target peptides. We continue to use X-ray crystallography to determine the structures of proteins of interest.

Publications:

Sheng, Y., Saridakis, V., Sarkari, F., Duan, S., Wu, T., Arrowsmith, C.H. and Frappier, L. 2006 Molecular recognition of p53 and Mdm2 by USP7/HAUSP. Nature Structural and Molecular Biology. 13, 285-291. )

Saridakis, V., Sheng, Y., Sarkari, F., Holowaty, M.N., Shire, K., Nguyen, T., Zhang, R.G., Liao, J., Lee, W., Edwards, A.M., Arrowsmith, C.H. and Frappier, L. 2005 Structure of the p53-binding domain of HAUSP/USP7 bound to Epstein-Barr Nuclear Antigen 1: Implications for EBV-mediate immortalization. Mol. Cell 18, 25-36.

Bochkarev, A., Bochkareva, E., Frappier, L. and Edwards, A.M. 1999 The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J. 18, 4498-4504.

Bochkarev, A, Bochkareva, E., Frappier, L. and Edwards, A.M. 1998 2.2 Å Structure of a permanganate-sensitive DNA site bound by the Epstein-Barr virus origin binding protein, EBNA1. J. Mol.. Biol. 284, 1273-1278.

Bochkarev, A., Pfuetzner, R., Edwards, A. and Frappier, L. 1997. Crystal structure of the single-stranded DNA binding domain of replication protein A bound to DNA. Nature 385, 176-181.

Bochkarev, A., Barwell, J., Pfuetzner, R., Bochkareva, E., Frappier, L. and Edwards, A. (1996) Crystal structure of the DNA binding domain of the Epstein-Barr virus origin binding protein, EBNA1, bound to DNA. Cell 84, 791-800.

Bochkarev, A., Barwell, J., Pfuetzner, R., Furey, W., Edwards, A. and Frappier, L. (1995) Crystal structure of the DNA binding domain of the Epstein-Barr virus origin binding protein, EBNA1. Cell 83, 39-46.