Domain substructure of HPV E6 oncoprotein: biophysical characterization of the E6 C-terminal DNA-binding domain.
E6 is a viral oncoprotein implicated in cervical cancers, produced by high-risk human papillomaviruses (HPVs). Structural data concerning this protein are scarce due to the difficulty of producing recombinant E6. Recently, we described the expression and purification of a stable, folded, and biologically active HPV16 E6 mutant called E6 6C/6S. Here, we analyzed the domain substructure of this mutated E6. Nonspecific proteolysis of full-length E6 6C/6S (158 residues) yielded N-terminal and C-terminal fragments encompassing residues 7-83 and 87-158, respectively. The C-terminal fragment of residues 87-158 was cloned, overexpressed, and purified at concentrations as high as 1 mM. The purified domain retains the selective four-way DNA junction recognition activity of the full-length E6 protein. Using UV absorption, UV fluorescence, circular dichroism, and nuclear magnetic resonance, we show that the peptide is primarily monomeric and folded with equal proportions of alpha-helix and beta-sheet secondary structure.
Nominé
Yves
Y
Laboratoire d'Immunotechnologie, UMR CNRS 7100, Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France.
Charbonnier
Sebastian
S
Ristriani
Tutik
T
Stier
Gunter
G
Masson
Murielle
M
Cavusoglu
Nukhet
N
Van Dorsselaer
Alain
A
Weiss
Etienne
E
Kieffer
Bruno
B
Travé
Gilles
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D027383:Papillomaviridae; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins