Targeting the Two Oncogenic Functional Sites of the HPV E6 Oncoprotein with a High-Affinity Bivalent Ligand

Abstract

The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, with both functions being crucial for HPV-induced oncogenesis. A chimeric protein was built by fusing a PDZ domain and an LxxLL motif, both known to bind E6. NMR spectroscopy, calorimetry and a mammalian protein complementation assay converged to show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all of the hrm-HPV E6 proteins tested but not to low-risk mucosal or cutaneous HPV E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspase 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers.

Keywords: antitumor agents; human papillomavirus; inhibitors; peptides; protein engineering.

Figure 1

Designing a chimeric bivalent ligand for hrm-HPV E6. a) Crystal structure of the E6/LxxLL complex.[7] The helical LxxLL motif of E6AP (grey) inserts into the LxxLL binding pocket of E6 (purple). The unbound C-terminal PBM of E6 (red) is extended and flexible. b) NMR structure of the MAGI1 PDZ2/E6 PBM complex.[9a] The E6 PBM (red) inserts into the peptide-binding groove of the PDZ domain (blue). c) Model structure of the PDZ-LxxLL chimera. d) Model structure of the chimera bound to E6. The long linker between folded PDZ core and LxxLL motif enables the simultaneous binding of PDZ and LxxLL to E6 PBM and E6 pocket, respectively.

Figure 2

NMR characterization of the PDZ-LxxLL chimera and its interaction with E6. Superimposed HSQC spectra (recorded at 25 °C) of E6-bound PDZ-LxxLL chimera (red) and free PDZ-LxxLL chimera (black). The black, blue, red, and green arrows indicate characteristic signals of free LxxLL, free PDZ, bound LxxLL, and bound PDZ, respectively, as identified by comparing different spectra (see Figures S3, S4).

Figure 3

Bivalency provides the chimera with a high affinity for hrm-HPV E6. a) Calorimetric titration of PDZ-LxxLL chimera (10 μM) with E6 (122 μM stock). b) Titration of E6 (50 μm) with LxxLL peptide (600 μm stock). c) Titration of the PDZ domain (10 μm) with E6 (122 μm stock). d) Thermodynamic profiles of E6/chimera, E6/LxxLL, and E6/PDZ interactions. e) Capture of endogeneous HPV18 E6 from Hela cells. A 6-his-GST-PDZ-LxxLL fusion was incubated with Hela cell extracts and double-purified over gluthatione and Ni-chelate affinity columns. Western blotting (WB) with anti-E6 antibody (right) specifically revealed a strong band at the size of E6 (17 kDa). f) Probing the binding of the chimera, PDZ, and LxxLL to 17 distinct HPV E6 proteins by using mammalian GPCA. Red and black numbers indicate NLR values above or below the lower threshold value for high-confidence binding (NLR=3.5), respectively (see Table S2 for source data).

Figure 4

Adenoviral (Ad) expression of the chimera specifically kills HPV-positive cells. a) U20S (HPV−), SiHa (HPV16+), and Hela (HPV18+) cells were transduced for 48 h with adenoviruses expressing GFP, myc-tagged PDZ, or myc-tagged PDZ-LxxLL chimera. The expression of P53, p21, PDZ, and the chimera was analyzed by western blotting with appropriate monoclonal antibodies. P53 and P21 levels specifically increased in the SiHa and HeLa cells transduced with PDZ and the chimera. b) Cells were transduced for 72 h with AdGFP, AdPDZ, and AdChimera. Crystal violet staining reveals massive cell death of HeLa and SiHa cells expressing the chimera and (to a lesser extent) PDZ.