Surface plasmon resonance analysis of the binding of high-risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI-1.
Fiche publication
Date publication
juillet 2011
Auteurs
Membres identifiés du Cancéropôle Est :
Dr KIEFFER Bruno, Dr MASSON Murielle, Pr ORFANOUDAKIS Georges, Dr TRAVE Gilles
Tous les auteurs :
Fournane S, Charbonnier S, Chapelle A, Kieffer B, Orfanoudakis G, Trave G, Masson M, Nomine Y
Lien Pubmed
Résumé
The E6 oncoproteins from high-risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain-containing proteins. Human MAGI-1 is a multi-PDZ domain protein implicated into protein complex assembly at cell-cell contacts. High-risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI-1 via a C-terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione-S-transferase (GST). This approach was applied to measure the binding of MAGI-1 PDZ1 to the C-termini of viral or cellular proteins. Both high-risk mucosal HPV E6 C-terminal peptides and cellular partners of MAGI-1 PDZ1 bind to MAGI-1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI-1 PDZ1 shows a preference for C-termini with a valine at position 0 and a negative charge at position -3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site-directed mutagenesis of the HPV16 C-terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ-binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K(499) residue of MAGI-1 as a novel determinant of binding specificity. Finally, we showed that MAGI-1 PDZ1 also binds to the C-termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI-1.
Référence
J Mol Recognit. 2011 Jul-Aug;24(4):511-23