Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance.
Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.
Zanier
Katia
K
UMR7100, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France. zanier@titus.u-strasbg.fr
Charbonnier
Sebastian
S
Baltzinger
Mireille
M
Nominé
Yves
Y
Altschuh
Danièle
D
Travé
Gilles
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D027383:Papillomaviridae; D011485:Protein Binding; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D009856:Oncogene Proteins, Viral; D044767:Ubiquitin-Protein Ligases