Structures of a human papillomavirus (HPV) E6 polypeptide bound to MAGUK proteins: mechanisms of targeting tumor suppressors by a high-risk HPV oncoprotein

Abstract

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.

FIG. 1.

Structures of three PDZ domains bound to the C-terminal peptide of HPV18 E6. (A) MAGI-1 PDZ1-E6 peptide complex (PDB identification [ID] no. 2I04). (B) SAP97/Dlg PDZ2-E6 peptide complex (PDB ID no. 2I0L). (C) SAP97/Dlg PDZ3-E6 peptide complex (PDB ID no. 2I0I). The PDZ structure is drawn as ribbons. The electron density corresponding to the E6 peptide was calculated before the peptide was built in (at a contour level of 1σ). A stick model of the bound peptide (shown as yellow sticks) fits nicely into the density in each complex. Clear electron density maps are seen for the side chains of six peptide residues in MAGI-1 PDZ1 and SAP97/Dlg PDZ3 complexes, while side chains of five residues are identified in the SAP97/Dlg PDZ2-peptide complex. (D) Consensus of the PDZ-binding ligand and the E6 peptide sequence used in this work. The position assignment (positions -1, -2, -3, etc.) is labeled below each amino acid.

FIG. 2.

Detailed interactions of the E6 peptide with the PDZ domains. (A) MAGI-1 PDZ1-peptide complex. The GFGF motif (G461 to F464) is labeled. Dashed lines denote hydrogen bonds. Blue spheres represent water molecules. (B) SAP97/Dlg PDZ2-peptide complex. The GLGF motif (G327 to F330) is labeled. (C) SAP97/Dlg PDZ3-E6 peptide complex. The GLGF motif (G474 to F477) is labeled. The E6 peptide is shown as a yellow stick in each complex. The involvement of the BC loop in E6 peptide binding is shown in each figure. (D) A close-up view of the interactions of R-6 with Q477 and the main chain carbonyl of MAGI-1 PDZ1. (E) Importance of the R-5 residue of E6 for the degradation of MAGI-1 and SAP97/Dlg. As shown above, R-5 contacts MAGI PDZ1 and hDlg/SAP97 PDZ2 through both the main chain and the side chain (also see Fig. 4D). In vitro-translated radiolabeled SAP97/Dlg, MAGI-1, and p53 proteins were incubated at 30°C alone (lane 2), with wild-type HPV18 E6 (lane 3), or with mutant (mut) HPV18 E6 that has R-5 mutated to G (R154G) (lane 4). The proteins in the assays were incubated for the times previously shown to give optimal degradation of the protein (2 h for SAP97/Dlg, 0.5 h for MAGI-1, and 1 h for p53). Note that the incubation time for MAGI-1 is four times shorter than for SAP97/Dlg, as the E6-mediated degradation for MAGI-1 is approximately four times faster. The input level of each target protein is shown in lane 1, and 20% of the input E6 protein is shown in the right-hand panel. (F) Reduced binding of the E6 R154G mutant to Dlg/SAP97 and MAGI-1 PDZ1. Equal amounts of in vitro-translated, radiolabeled wild-type or mutant E6 (R154G) protein were incubated with GST alone, GST-Dlg, or GST-M1P1 (MAGI-1 PDZ domain 1). The autoradiograph in the upper panel shows the amount of radiolabeled E6 protein retained, with 20% of the input in the right-hand panel. The lower panel shows the GST protein inputs, stained with Coomassie; the GST proteins are indicated with arrows. (G) A histogram showing the quantified results of the assays exemplified in panel F, determined by phosphorimaging analysis and expressed as increases (n-fold) of binding above binding to GST alone. Standard deviations are shown.

FIG. 3.

(A) Sequence alignment of MAGI-1 PDZ1 with SAP97/Dlg PDZ2 and PDZ3. Important secondary structural elements are highlighted; two key residues belonging to the BC loop, D470 of MAGI-1 PDZ1 and N338 of SAP97/Dlg PDZ2, are marked by * and #, respectively. (B) Sequence alignment of the C-terminal sequences from nine high-risk HPV types. The position number (-1, -2, -3, etc.) used for each residue is listed above the alignment.

FIG. 4.

Different interactions between E6 peptide residues Q-2 and R-6 with PDZs. (A) An overview of the superposition of MAGI-1 PDZ1 (silver brown) and SAP97/Dlg PDZ2 (green) on PDZ3 (salmon pink). (B) A close-up view of the marked box in panel A, showing the alternative orientations of Q-2 of the three E6 peptides bound to the three PDZs. Residues are in yellow for the MAGI-1 PDZ1-peptide complex, in green for the SAP97/Dlg PDZ2 complex, and in magenta for the SAP97/Dlg PDZ3 complex. (C) A close-up view of the area showing that the hydrogen bond between R-6 and Q477 in MAGI-1 PDZ1 with the E6 peptide complex would be obstructed in the SAP97/Dlg PDZ2-peptide complex by the presence of the longer BC loop and H340 of the PDZ2 domain. (D) Summary of the interactions of each residue of the HPV18 E6 peptide in the three PDZ complexes. mc, main chain interaction; sc, side chain interaction; mc&sc, interaction of both the main chain and the side chain.

FIG. 5.

Dimer formation of MAGI-I PDZ1. (A) An overview of the MAGI-1 PDZ1 homodimer, shown in a ribbon diagram. Each monomer is shown in a different color (salmon or blue). The residues involved in contacts at the interface are shown as sticks. (B) A close-up view of the interactions at the dimer interface: two disulfide bridges are shown as green sticks, hydrogen bonds as dashed lines, and water molecules as blue spheres. Hydrophobic residues were found to cluster at the core of the interface. (C) Direct interaction of R-6 with D473 of the bottom monomer (salmon). This R-6 reaches across the dimer interface from the E6 peptide bound to the substrate groove of the top monomer (in blue). (D) Dimer formation of MAGI-1 PDZ1 in solution. About 10 μl of purified MAGI-1 PDZ1 (3.0 μg/μl) was treated with 10 μl SDS loading buffer in the absence or presence of 10 mM DTT (lanes 2 and 3), followed by a 12.5% SDS-PAGE analysis. Purified SAP97/Dlg PDZ3 was used as a control (lanes 4 and 5). The dimer form was detected only for MAGI-1 PDZ1 under nonreducing conditions (without DTT) (lane 2). All the PDZ domains run as a relatively diffused band as monomers, possibly due to their small size (∼8 kDa).