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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

Authors

  • Sadek Fournane,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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    • These authors contributed equally to this work.

  • Sebastian Charbonnier,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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    • These authors contributed equally to this work.

  • Anne Chapelle,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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  • Bruno Kieffer,

    1. Biomolecular NMR Group, UMR CNRS 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Rue Laurent Fries, 67400 Illkirch Cedex, France
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  • Georges Orfanoudakis,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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  • Gilles Travé,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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  • Murielle Masson,

    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
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  • Yves Nominé

    Corresponding author
    1. Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France
    • Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France.
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Abstract

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 K499 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. Copyright © 2010 John Wiley & Sons, Ltd.

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