Purification and biophysical characterization of a minimal functional domain and of an N-terminal Zn2+-binding fragment from the human papillomavirus type 16 E6 protein

The E6 Zn2+-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2-142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143-151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn2+ ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2-77, E6-N) was similarly purified. Unlike E6(Delta 143-151), E6-N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn2+ ion by electrospray mass spectrometry and by atomic absorption analysis. UV-visible spectroscopic analysis of Co2+-substituted E6-N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6-N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn2+ binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143-151), at low concentrations, but dimerization occurs at high concentrations (Deltad 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143-151) and E6-N. The results support a model of monomeric E6 possessing two functionally critical Zn2+-binding motifs.