1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6. - Archive ouverte HAL Access content directly
Journal Articles Journal of Biomolecular NMR Year : 2005

1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6.

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Laurent Miguet
  • Function : Author
Noelle Potier
Alain van Dorsselaer
  • Function : Author
Robert Andrew Atkinson
  • Function : Author

Abstract

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.

Dates and versions

hal-00187709 , version 1 (15-11-2007)

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Cite

Yves Nominé, Sebastian Charbonnier, Laurent Miguet, Noelle Potier, Alain van Dorsselaer, et al.. 1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6.. Journal of Biomolecular NMR, 2005, 31 (2), pp.129-41. ⟨10.1007/s10858-004-7802-y⟩. ⟨hal-00187709⟩
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