Biophysical characterization of HPV-18 E6 high molecular weight oligomers in solutions

GARCIA ALAI, M.M; ALONSO, L.G; SMAL, C; PRAT GAY, G

Angra dos Reis, RJ, Brazil

Congreso; First Latin America Protein Society Meeting; 2004

High-risk HPVs such as HPV-18 is causally associated with human cervical cancer. HPV-18 E6 is a putative Zn binding protein of 158 amino acids that contains two copies of a Cys-X-X-Cys motive and a total of 10 cysteines. This oncoprotein has a key role in transformation for targeting the p53 tumor suppressor for degradation. Some other functions such as degradation of PDZ domain-containing proteins, four-way DNA binding, transcriptional activation and telomerase deregulation have been described but there is no structural information or biochemical characterization that could explain a precise molecular mechanism of action. Recombinant production of HPV E6 is known to be difficult due to formation of inclusion bodies and failure to obtain re-folded soluble protein. (Heterogeneous moieties and precipitation dependent on the oxido-reduction conditions were described leading to production of E6 fused to large globular domains for solubility enhancement or cysteine mutants.) In this current work we present a biophysical characterization of recombinant wild type HPV-18 E6 (Nt(6His)-E6 and non-fusion E6 protein) whose behavior in solution explains the notorious reputation achieved by this protein for its production and purification. We show that wild type HPV-18 E6 forms soluble high weight oligomers as has been previously described for the other HPV oncoprotein E7 (Biochemistry, 43(12): 3310-7). These oligomers are excluded from a 10 to 300 Kda size exclusion chromatography and seen as spherical particles of 40 nm average diameter by atomic force microscopy. E6 oligomers are sensible to pH and salt concentration, presenting sharp transitions followed by circular dichroism and light scattering. They are highly stable to heat denaturation and once assembled undergo inter molecular oxidation that stabilize the macromolecular structure