Solution Structure of the hDlg/SAP97 PDZ2 Domain and Its Mechanism of Interaction with HPV-18 Papillomavirus E6 Protein^,

Abstract

The E6 protein from high-risk types of human papillomavirus (HPV) binds PDZ-domain containing proteins and targets them for degradation. We used isothermal titration calorimetry to measure the interaction of a peptide from the C-terminus of HPV-18 E6 to the second PDZ domain (PDZ2) from the human homologue of the Drosophila discs large tumor suppressor protein (hDlg). Isothermal titration calorimetry experiments with a series of peptides showed that HPV-18 E6 bound hDlg PDZ2 about 5-fold stronger than HPV-16 E6, that the contribution of Arg154 to binding was about 1 kcal/mol, and that the binding was disabled by phosphorylation at Thr156. We then used NMR to determine the solution structure of the complex of PDZ2 bound to the HPV-18 E6 peptide. The resultant structures were of high quality and had backbone root-mean-square deviations of less than 0.5 A. The structure shows a novel mode of interaction in which six residues of the HPV-18 E6 peptide are contacted by the PDZ2 domain, in contrast to the typical four residues used by class I PDZ domains. Molecular dynamics simulations supported a model in which the C- and N-terminal ends of the peptide had different mobilities within the complex. Comparison of the NMR complex structure to previously determined X-ray structures of PDZ2 by itself and bound to different peptides allows a description of conformational changes required for PDZ2 to bind to HPV-18 E6.

A Systematic Analysis of Human Papillomavirus (HPV) E6 PDZ Substrates Identifies MAGI-1 as a Major Target of HPV Type 16 (HPV-16) and HPV-18 Whose Loss Accompanies Disruption of Tight Junctions

“…We know from previous studies that minor differences in the PDZ-binding motif between HPV type 16 (HPV-16) and HPV-18 E6 can significantly affect PDZ domain targeting, with HPV-18 E6 preferentially binding to hDlg, while HPV-16 E6 preferentially binds to hScrib (47). Similar results have also been reported for CAL (19), and structural studies on E6 complexed with Dlg and MAGI-1 provide some molecular explanations for these apparent differences (29,44,54). It is also clear, however, that while PDZ selection by E6 is highly specific, it can give potentially misleading results if assessed only under conditions of overexpression or in vitro, since at high concentrations the E6 PBM can potentially recognize any class I PDZ domain (54).…”

Sorting of β1-Adrenergic Receptors Is Mediated by Pathways That Are Either Dependent on or Independent of Type I PDZ, Protein Kinase A (PKA), and SAP97

“…Based upon these studies, the functional residues in type I PDZs were the hydrophobic residue at P ϭ 0 and Ser/Thr at P ϭ Ϫ2 (5, 31). These results were in agreement with crystal structural data between the type I PDZ of GluR1 (ATGL) or the E6 protein of human papillomavirus-18 (ETQV) with PDZ2 of SAP97 (21,22). These complexes showed strong interactions between the hydrophobic amino acid at P ϭ 0 and the ␤B domain of PDZ2 A, HEK-293 cells expressing the indicated FLAG-tagged receptors were exposed to 10 M isoproterenol for 0, 3, or 6 h. Total cell extracts were probed by Western blotting with anti-FLAG IgG to compare the levels of ␤ 1 -AR chimeras and with anti-␤-actin to equalize the amounts of loaded protein lysates.…”

Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains

“…Three different and well-studied PDZ domains were included in the study, SAP97 PDZ2, PTP-BL PDZ2 and PSD-95 PDZ3. For each of these PDZ domains, we selected a wild-type peptide, based on previous work 32, [34][35][36][37][38] (see legend to Fig. 2 for wild-type and mutant peptides).…”