Structural analysis of the human papillomavirus type 16-E2 transactivator with antipeptide antibodies reveals a high mobility region linking the transactivation and the DNA-binding domains.
In order to probe the structure of the transcription factor encoded by the E2 Open Reading Frame of papillomaviruses, we raised polyclonal antibodies against a series of synthetic peptides that cover the HPV16-E2 protein. In gel shift experiments with the native form of the protein, we detected supershifts (caused by the binding of antibodies to the E2-DNA complex) with antibodies synthesized against peptides covering a central region 50 residues long in the E2 protein. On the contrary, antibodies raised against peptides from the NH2- and COOH-termini did not give any supershifted band. Western blot experiments showed that several of these non reacting antibodies did however interact with the denatured protein. These results suggest that the central region that connects the NH2-terminal domain responsible for transcriptional activation and the COOH-domain involved in DNA-binding is exposed and maintained in a conformation resembling the peptide, indicating a high mobility region. In contrast, the DNA-binding and transactivation domains were not recognized by the antipeptide antibodies, in line with secondary structure predictions and sequence comparisons indicating that the E2 protein consists of structured and conserved NH2 and COOH-terminal regions separated by a non-conserved and unstructured region. This flexible 'hinge' region may facilitate contacts between E2 dimers at distance in mechanisms of transcriptional activation steps that involve homosynergy or DNA-looping.
Gauthier
J M
JM
Département des Biotechnologies, Institut Pasteur, Paris, France.
Dillner
J
J
Yaniv
M
M
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry*; D027383:Papillomaviridae / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D016415:Sequence Alignment; D015534:Trans-Activators / Q000737:chemistry*
D000906:Antibodies; D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D010446:Peptide Fragments; D015534:Trans-Activators; D011505:Protein-Tyrosine Kinases