Specific N-methylations of HPV-16 E7 peptides alter binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type 16 E7 protein (HPV-16 E7) and the retinoblastoma growth suppressor protein (RB) is believed to contribute to the process of cellular transformation that leads to cervical carcinoma. Genetic analysis of the HPV-16 E7 protein has shown that the segment of E7 homologous to the conserved region 2 of adenovirus 5 E1A protein is involved in both RB binding and E7-mediated cell transformation. We have previously shown that a peptide colinear with HPV-16 E7 residues 21-29 was able to block immobilized species of E7 from binding to RB protein. The current study reports the effects of different chemical modifications of this peptide. One type of modification, methylation of the alpha-amino nitrogens contributed by Leu22, Tyr25, and Leu28, resulted in a 45-fold increase in E7/RB binding antagonist activity. This increased antagonist activity is sequence-specific since methylation of the amino groups contributed by Tyr23, Cys24, or Glu26 resulted in a profound loss of binding antagonist activity. Using a newly developed binding assay we determined that the apparent dissociation constant for recombinant HPV-16 E7 protein binding to recombinant human RB protein is 1.3 nM. The peptide Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7 amide was determined to be a competitive inhibitor of HPV-16 E7 binding to RB with a Ki value of 32 nM.