SPR based kinetic analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) Sensorgrams obtained by injecting purified F47R 4C/4S E6 at the indicated concentrations on a surface capturing E6APpep. RU stands for SPR response units. Color lines represent analyte protein injections at the indicated concentrations. (<i>B</i>) Interaction of E6 F47R 4C/4S with surface-captured pep11**. Color and black lines indicate analyte injections and global fits to a 1∶1 binding model, respectively.</p

NMR interface analyses of the HPV16 E6/E6APpep and HPV16/pep11** interactions.

<p>Indicated are the changes in the intensities of backbone amide cross-peaks of E6 F47R 4C/4S residues upon addition of a 1-fold excess of unlabeled E6APpep (<i>A</i>) or pep11** (<i>B</i>). I_bound/I_ref thresholds of 0.7 and 0.6 are indicated by colored horizontal lines (light green/dark green and pink/purple for the E6APpep and pep11** interactions, respectively). Error bars (I_noise/I_ref ) report on the signal-to-noise (S/N) ratio in the reference spectra (see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112514#pone-0112514-g002" target="_blank">Figure 2</a>). Amide cross-peaks with a I_noise/I_ref >0.25 are considered noisy peaks. Cyan shaded areas indicate unassigned residues or prolines (which are additionally highlighted by yellow dots). HPV16 E6 secondary structure elements deduced from the HPV16 E6/E6APpep x-ray structure are indicated above the histograms.</p

Intracellular analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) Mammalian two-hybrid analyses in HeLa cells expressing individual peptides, as indicated, linked to GAL4-BD, and wt HPV16 E6 linked to the VP16-AD. pep11**m does not bind to HPV16 E6 and served as a negative control. Shown are relative activities of the co-transfected luciferase reporter plasmid under transcriptional control of GAL4-binding sites above those of control-transfected cells (expressing corresponding peptide-GAL4-BD fusions together with empty control vector pACT; values arbitrarily set at 1.0). Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above horizontal lines indicate statistically significant differences from pACT-transfected cells, with p-values of ≤0.001 (***). (<i>B</i>) and (<i>C</i>) Mutational analyses of HPV16 E6 to identify amino acid residues that contribute to E6APpep (<i>B</i>) or pep11** (<i>C</i>) binding. The luciferase signal for the interaction of wt HPV16 E6 with E6APpep or pep11** was set at 100% (left columns in (<i>B</i>) and (<i>C</i>), respectively). The horizontal line indicates 1.5-fold inhibition. Individual amino acid exchanges are indicated below each column. Error bars indicate standard deviation values. (<i>D</i>) Statistical analysis of the differential binding behaviour of HPV16 E6 mutants 16E6R55A and 16E6Y70A. The values for the interaction between wt HPV16 E6 protein with E6APpep or pep11**, respectively, were set at 100%. Asterisks above the horizontal lines indicate statistically significant reductions of luciferase activities, with p-values of ≤0.01 (**) or ≤0.001 (***). Error bars indicate standard deviation values.</p

NMR analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) ¹H,¹⁵N SOFAST-HMQC spectra of 100 µM ¹⁵N E6 F47R 4C/4S samples in the absence (black spectra) and presence of 1∶1 stoichiometric ratios (cyan and red spectra) of unlabeled E6APpep (upper panel) or pep11** (lower panel). Cyan spectra were recorded immediately after peptide addition (t = 0 h), whereas red spectra were recorded after 3 h of incubation in the presence of the peptide. Note that in the case of spectra of E6/pep11** samples some signals have lower intensity in the red spectrum than in the cyan spectrum (see enlarged view of lower panel). This decrease in signal intensity is concomitant with the appearance of a white precipitate of E6 in the NMR tube. (<i>B</i>) pep11** induced E6 aggregation monitored by the decrease of the intensity of the W132 indole cross-peak. Intensity changes are expressed as a I_bound/I_ref ratio, where I_bound is the cross-peak intensity in the peptide-bound spectra and I_ref the cross-peak intensity in the reference (unbound) spectrum. W132 indole intensities were derived from ¹H,¹⁵N SOFAST-HMQC spectra recorded at regular time intervals on three different samples containing ¹⁵N labeled E6 F47R 4C/4S and unlabeled pep11** mixtures with concentration ratios adjusted to 1∶0.5 (gray diamonds), 1∶1 (cyan squares) and 1∶2 (green triangles). E6 concentrations in all three samples were adjusted to 100 µM. X-axis error bars correspond to the duration of the NMR experiment (i.e. error bar: 15 min), whereas y-axis error bars report on the signal-to-noise (S/N) ratio in the reference spectra, which, in this case, is expressed as the inverse of the S/N for sake of clarity (i.e. error bar: ± I_noise/I_ref). Estimates of the noise were obtained using the program NMRpipe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112514#pone.0112514-Delaglio1" target="_blank">[32]</a>.</p

E6-binding peptides mediating intracellular formation of a trimeric complex with HPV16 E6 and p53.

<p>(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: HPV16 E6 is linked to GAL4-BD, p53 is linked to VP16-AD, individual peptides are expressed from a co-transfected expression vector, in fusion with hrGFP. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Expression of HPV16 E6-GAL4-BD with p53-VP16-AD, together with hrGFP-linked peptides, as indicated, or, as negative control, hrGFP alone. Shown are relative luciferase activities (RLA) above those of control-transfected cells, expressing HPV16 E6-GAL4-BD fusions together with the co-transfected basic vectors pACT and pCEP4hrGFP; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing HPV16 E6-GAL4-BD together with p53-VP16-AD, in the absence of the peptides (co-transfected basic vector pCEP4hrGFP), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of individual peptides linked to hrGFP. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.</p

Immunoblot analyses of HPV16 E6, upon ectopic co-expression with E6-targeting peptides.

<p>Expression of HPV16 E6 in HeLa cells together with either hrGFP-linked control peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent). Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.</p

Trimeric complex formation between HPV16 E6, p53 and pep11** or E6AP, in GST pull-down analyses.

<p>(A) p53 was expressed in H1299/K3 cells and pull-down assays were performed with GST-tagged HPV16 E6 (GST-16E6) in the presence of no peptide, synthetic pep11** or synthetic pep11**m. E6AP and p53 bound to GST-16 E6 are indicated. (B) The H1299/K3 lysate was pre-cleared from endogenous E6AP protein by pre-incubation with GST-16 E6. E6AP and p53 bound to GST-16 E6 are indicated.</p

HPV16 E6 mediating intracellular formation of a trimeric complex with E6-binding peptides and p53.

<p>(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: Individual peptides are linked to GAL4-BD, p53 is linked to VP16-AD, E6 proteins are expressed from a co-transfected expression vector. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Co-expression of individual peptides linked to the GAL4-BD, as indicated, together with p53-VP16-AD and HPV16 E6 or HPV 11 E6, respectively. Empty expression vector pNCMV served as negative control. Indicated are relative luciferase activities (RLA) above those of control-transfected cells, expressing the corresponding peptide-GAL4-BD fusions together with the co-transfected empty vectors pACT and pNCMV; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing the corresponding peptides linked to GAL4-BD together with p53-VP16-AD, in the absence of E6 (co-transfected empty vector pNCMV), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of Flag-tagged HPV16 E6 and HPV11 E6 and of (D) individual peptides linked to GAL4-DB. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.</p

Intracellular co-localization of pep11** and HPV16 E6.

<p>(A) mCherry-HPV16 E6 was co-expressed in HeLa cells with either hrGFPpep11** (upper panel) or hrGFP-pep11**m (lower panel). (B) Immunostaining for hrGFP upon co-expression of mCherry-HPV16 E6 with hrGFPpep11** in HeLa cells. (C) Upper panel: Nocodazole treatment of HeLa cells upon co-expression of mCherry-HPV16 E6 with hrGFPpep11**. Lower panel: Untreated control cells. (D) Immunostaining for vimentin in HeLa cells co-expressing mCherry-HPV16 E6 with either hrGFPpep11** (upper panel) or hrGFP-pep11**m (lower panel).</p

Immunoblot analyses of endogenous HPV16 E6, upon intracellular expression of E6-targeting peptides.

<p>Expression of either hrGFP-linked peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent) in HPV-16 positive MRI-H186 cells. Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by the activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP, of p53, and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.</p