Biophysical characterization of HPV-18 E6 High Molecular Weight Oligomers in solution.

GARCÍA ALAI, M.M., ALONSO, L.G., SMAL, C. AND PRAT GAY, G. DE

Angra dos Reis, R.J., Brasil, 8-12 de noviembre de 2004.

Congreso; First Latin American Protein Society Meeting; 2004

The Protein Society

Biophysical characterization of HPV-18 E6 High Molecular Weight Oligomers in solution. María Marta García Alai, Leonardo G. Alonso, Clara Smal and Gonzalo de Prat Gay. Fundación Instituto Leloir, Av. Patricias Argentinas 435, (1405) Buenos Aires, Argentina. 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 cys. E6 has a key role in transformation by 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 to be associated to its malignity activity. Despite of this 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. In this 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 molecular weight oligomers. 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 stabilizes the macromolecular structure.