These authors contributed equally to this work.
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
Article first published online: 14 SEP 2010
Copyright © 2010 John Wiley & Sons, Ltd.
Journal of Molecular Recognition
Volume 24, Issue 4, pages 511–523, July/August 2011
How to Cite
Fournane, S., Charbonnier, S., Chapelle, A., Kieffer, B., Orfanoudakis, G., Travé, G., Masson, M. and Nominé, Y. (2011), 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. J. Mol. Recognit., 24: 511–523. doi: 10.1002/jmr.1056
- Issue published online: 7 APR 2011
- Article first published online: 14 SEP 2010
- Manuscript Accepted: 25 APR 2010
- Manuscript Revised: 22 APR 2010
- Manuscript Received: 23 FEB 2010
- PDZ domain;
- HPV E6;
- protein/peptide interaction;
- medium throughput screening
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.