Source:http://linkedlifedata.com/resource/pubmed/id/20842623
General Info
Affiliation
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.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 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.
PMID
20842623
Publication types
Research Support, Non-U.S. Gov't