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.
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.
Fournane
Sadek
S
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.
Charbonnier
Sebastian
S
Chapelle
Anne
A
Kieffer
Bruno
B
Orfanoudakis
Georges
G
Travé
Gilles
G
Masson
Murielle
M
Nominé
Yves
Y
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051528:Guanylate Kinases; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D051528:Guanylate Kinases; C113338:MAGI1 protein, human