Biophysical characterization of the interaction domains and mapping of the contact residues in the XPF-ERCC1 complex.
XPF and ERCC1 exist as a heterodimer to be stable and active in cells and catalyze DNA cleavage on the 5'-side of a lesion during nucleotide excision repair. To characterize the specific interaction between XPF and ERCC1, we expressed the human ERCC1 binding domain of XPF (XPF-EB) and the XPF binding domain of ERCC1 (ERCC1-FB) in Escherichia coli. Milligram quantities of a heterodimer were characterized with gel filtration chromatography, an Ni(2+)-NTA binding assay, and analytical ultracentrifugation. Cross-linking experiments at high salt concentrations revealed that XPF interacts with ERCC1 mainly through hydrophobic interactions. XPF-EB was also shown to homodimerize in the absence of ERCC1. NMR cross-saturation methods were applied to map the residues involved in formation of the XPF-EB.XPF-EB homodimer and the XPF-EB.ERCC1-FB heterodimer. Helix H3 and the C-terminal region of XPF-EB were either within or in close proximity to the homodimer interface, whereas the ERCC1-FB binding site of XPF-EB was distributed across helix H1, a small part of H2, H3, and the C-terminal region, most of which exhibited large changes in chemical shift upon ERCC1 binding. The XPF-EB heterodimeric interface is larger than the XPF-EB homodimeric one, which could explain why XPF has a stronger affinity for ERCC1 than for a second molecule of XPF. The XPF binding sites of ERCC1 were located in helices H1 and H3 and in the C-terminal region, similar to the involved surface of XPF. We used cross-saturation data and the crystal structure of related proteins to model the two complexes.
Choi
Yun-Jeong
YJ
Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejon 305-701, Korea.
Ryu
Kyoung-Seok
KS
Ko
Yun-Mi
YM
Chae
Young-Kee
YK
Pelton
Jeffrey G
JG
Wemmer
David E
DE
Choi
Byong-Seok
BS
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C095888:xeroderma pigmentosum group F protein; C071447:ERCC1 protein, human; D004720:Endonucleases