The Zn binding domain of E6 protein from HPV is rebuilt from its splice product, E6*.

ANGELES HEER Y GONZALO PRAT GAY

Punta Ballena

Simposio; Thiol metabolism and redox regulation of cellular functions; 2011

E6*I is the most representative E6 spliced mRNA in HPV transformed cells, cervical cancer cell lines and clinicalsamples and encode for the first 50 amino acid of the E6 oncoprotein, including only half of the first Zn binding motif ofthe full length E6 protein.In this context, we want to characterize the E6*I solution behavior in terms of its folding, stability and Zn bindingproperties.Depending on the solution pH, E6*I is able to adopt multiple conformations, including species enriched in alpha-helixor a β-sheet secondary structure. As many small peptides, E6*I is prone to assemble into high molecular weight solublestructures. At low pH, E6*I shares properties with amyloid-like structures as judged by its ability to bind Thioflavine T.At pH near to neutrality, assembles into oligomers with high alpha-helix content. Interestingly, addition of Zn precludesthe oligomerzation of the sample and maintains the protein in a dimeric non-aggregated form.We show that E6*I dimerizes in the presence of Zn to form a cysteine coordinating zinc high affinity center, rebuildingthe zinc finger domain present in full length E6. Metal binding inhibits the oxidation of two of its three cysteins thatotherwise oxidize to form an intramolecular disulfide bridge.Preliminary experiments with the C-terminal portion of E6*I which contain the cysteines involved in Zn bindingindicates, however, that the N-terminal portion is neccesary to maintain the high affinity for the metal atom. Zn bindingprecludes the alpha-helix formation in TFE titration experiments regulating the conformational state of the polypetide.We suggest that the high Zn binding affinity and its conformational properties are a hallmark feature to achieve itsbiological function.