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Title
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1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6.
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Authors
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Y.Nominé,
S.Charbonnier,
L.Miguet,
N.Potier,
A.Van Dorsselaer,
R.A.Atkinson,
G.Travé,
B.Kieffer.
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Ref.
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J Biomol Nmr, 2005,
31,
129-141.
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PubMed id
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Abstract
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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.
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