Solution structure determination and mutational analysis of the papillomavirus E6 interacting peptide of E6AP

E6AP is a cellular protein that binds cancer-related papillomaviral E6 prot eins. The E6 binding domain, called E6ap, is located on an 18-amino acid se gment of E6AP. The corresponding peptide was synthesized and its structure determined by nuclear magnetic resonance spectroscopy. The overall structur e of the peptide is helical. A consensus E6-binding sequence among differen t E6 interacting proteins contains three conserved hydrophobic residues. In the structure of the E6AP peptide, the three conserved leucines (Leu 9, Le u 12, and Leu 13) form a hydrophobic patch on one face of the cr-helix. Sub stitution of any of these leucines with alanine abolished binding to E6 pro tein, indicating that the entire hydrophobic patch is necessary. Mutation o f a glutamate to proline, but not alanine, also disrupted the interaction b etween E6 and E6AP protein, suggesting that the E6-binding motif of the E6A P protein must be helical when bound to E6. Comparison of the E6ap structur e and mutational results with those of another E6-binding protein (E6BP/ERC -55) indicates the existence of a general EG-binding motif.