1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6.
E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced 15N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most 1H and 15N resonances and identified the elements of secondary structure of the domain. The domain displays an original alpha/beta topology with roughly equal proportions of alpha-helix and beta-sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by 1H,15N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by 15N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with mus-ms time scale motions occurring in localised regions of the monomeric domain.
Nominé
Yves
Y
Laboratoire d'Immunotechnologie, CNRS UMR 7100, Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France.
Charbonnier
Sebastian
S
Miguet
Laurent
L
Potier
Noelle
N
Van Dorsselaer
Alain
A
Atkinson
R Andrew
RA
Travé
Gilles
G
Kieffer
Bruno
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000378:metabolism
C068651:E6 protein, Human papillomavirus type 16; D009587:Nitrogen Isotopes; D009856:Oncogene Proteins, Viral; D011522:Protons; D012097:Repressor Proteins; D015032:Zinc