E6 protein of human papillomavirus type 18 binds zinc.
The E6 open reading frames of human and animal papillomaviruses encode a transforming protein containing a conserved pattern of repeating cysteine doublets (cys-x-x-cys) similar to that found in steroid receptor zinc finger proteins. The spacing between the cysteine doublets, however, is twice as long as in any other zinc finger protein. To demonstrate that an E6 protein could indeed bind zinc, we synthesized the human papillomavirus type 18 E6 protein in insect cells with a baculovirus vector and analysed the protein for zinc-binding activity by a zinc-blot assay. Probing of E6 protein blotted to nitrocellulose from SDS-polyacrylamide gels with radioactive [65Zn]Cl2 demonstrated that it possessed zinc-binding activity. Reduction of cysteines with DTT prior to the addition of zinc greatly increased the zinc-binding activity of blotted E6 protein. Competition experiments showed that Cd2+ and Co2+ were more effective competitors for zinc-binding than Mg2+ or Ca2+, indicating that zinc atoms may be tetrahedrally coordinated in E6-zinc complexes. We mapped zinc-binding protein domains by proteolytic cleavage and demonstrated that a small 4kDa fragment of the protein retained zinc-binding activity, consistent with a model of individual 29 amino acid zinc-binding 'fingers'.
Grossman
S R
SR
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Laimins
L A
LA
D001667:Binding, Competitive; D004268:DNA-Binding Proteins; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D015032:Zinc; D004229:Dithiothreitol