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 α/β topology with roughly equal proportions of α-helix and β-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 μs–ms time scale motions occurring in localised regions of the monomeric domain.
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Nominé, Y., Charbonnier, S., Miguet, L. et al. 1H and 15N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6. J Biomol NMR 31, 129–141 (2005). https://doi.org/10.1007/s10858-004-7802-y
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DOI: https://doi.org/10.1007/s10858-004-7802-y