Human papillomavirus type 18 E7 protein requires intact Cys-X-X-Cys motifs for zinc binding, dimerization, and transformation but not for Rb binding.
Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.
McIntyre
M C
MC
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Frattini
M G
MG
Grossman
S R
SR
Laimins
L A
LA
D017500:Adenovirus E1A Proteins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006207:Half-Life; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016160:Retinoblastoma Protein / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D004492:Edetic Acid; D015032:Zinc; D003545:Cysteine; D012413:Rubidium