51Z2 derived E6CT6 peptide versus E6CT11 peptide binding to hDlgPDZ2.

<p>SPR data. <b>A</b> E6CT6, <b>B</b> E6CT11. Sensorgrams of E6CT6 and E6CT11 binding injected in triplicate (black lines) are shown overlaid with the best fit derived from a 1∶1 interaction model including a mass transport term (orange lines). Peptide concentrations of 3.125, 6.25, 12.5, 25, 50, 100 and 200 µM are shown. The binding parameters were obtained by kinetic analysis of association and dissociation phases (left panels) or by steady state analysis (right panels) utilizing signals of plateaus depicted in the corresponding left panel.</p

Interaction of E6CT11 with hDlgPDZ2.

<p><b>A</b> Combined ¹H and ¹⁵N chemical shift perturbation (as detailed in SI) of 100 µM hDlgPDZ2 in complex with 300 µM E6CT11 peptide versus 300 µM E6CT6 peptide. Residues without observable amide shifts are denoted with X. The inset of a region of the corresponding HSQC spectra show unperturbed as well as perturbed signals. Red contours: hDlgPDZ2 complexed with E6CT11, blue contours: hDlgPDZ2 complexed with E6CT6. Note that the side chain amide signals of Asn339 were also perturbed by more than 2× the average value. <b>B</b> Structure of the hDlgPDZ2-E6CT11 complex. The bundle of 20 best E6CT11 structures (residues 141 to 151, dark grey) is shown together with a ribbon of the closest-to-mean hDlgPDZ2 structure (hDlg residues 318–406). Peptide structures were fitted to residues 143 to 151 and the termini are indicated. Secondary structure elements are labeled. The boxed inset depicts per-residue backbone order parameters of the complexed E6CT11 peptide. <b>C</b> Details of the hDlgPDZ2-E6CT11 complex. hDlgPDZ2 backbone trace depicted in light gray. PDZ side chains (heavy atoms) of residues showing most perturbed combined amide group chemical shifts (backbone and Asn339 side chain; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone-0062584-g006" target="_blank">Figure 6a</a>) are depicted in green and labeled, while the closest-to-mean E6CT11 peptide structure (heavy atoms, residues 143–151) is presented in dark gray. <b>D</b> Schematic depiction of intermolecular hydrogen bonds and salt bridges in the clostest-to-mean complex structure. Indicated side-chains start at the Cβ atom. Hydrogen bonds are indicated as dashed lines. Secondary structure elements β* and β2 are emphasized by arrows; hDlgPDZ2 residues appear gray, while peptide residues are depicted in black.</p

Temperature sensitivity of the HPV 51 C-terminal zinc-binding domain 51Z2.

<p>Nine [¹H-¹⁵N]-HSQC spectra of an 51Z2 sample were recorded with identical spectral parameters and only the temperature was increased as indicated in the spectral plots. Residues that were still observable at 45°C are indicated in the 45°C spectrum. The assignment for the 45°C signals was based on the assignment for 10°C sample temperature (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone-0062584-g002" target="_blank">Figure 2</a>) by tracking peak positions with increasing temperatures. Tentative assignments which were not unambiguous are given in parentheses. One control shows the 51Z2 spectrum after returning to the start temperature.</p

Statistics for the 20 best 51Z2 and hDlgPDZ2-E6CT11 peptide complex structures.^a

a<p>Methods detailed in the Supplementary Information.</p>b<p>including the intermolecular (inter-chain) distance restraints.</p>c<p>Residue numbers 80 to 151 refer to position of the full-length HPV 51 E6 protein (UniProtKB entry: P26554), while residues 318 to 406 refer to positions of the full length hDlg protein (UniProtKB entry: Q12959). Flexible non-native residues (amino-terminal GSHM of 51Z2 and carboxy-terminal His₆-Tag of hDlgPDZ2) were not included in this structural statistics. For 51Z2 r.m.s.d. calculations include residues 80–151 or 80–140, respectively (see text). For the hDlgPDZ2-E6CT11 complex, r.m.s.d. calculations include residues 318–406 (hDlg) and 141–151 (E6) or 318–406 (hDlg) and 143–151 (E6), respectively.</p

Solution structure of the C-terminal zinc-binding domain of HPV 51 E6 (51Z2).

<p>The bound zinc ion is represented as grey sphere. <b>A</b> Stereo view of the 51Z2 bundle of the 20 structures with the lowest energy after CNS refinement. <b>B</b> Ribbon view of the structural ensemble as in <b>A</b> with labeled secondary structure elements. The less ordered C-terminal residues 141–151 are omitted for clarity. <b>C</b> represents the rotated ensemble of <b>B</b>. <b>D</b> Backbone hydrogen bonds between residues on β2 and β5 strands stabilize the arrangement of both 51Z2 β-sheets. Side chains omitted for clarity. <b>E</b> The bidentate H-bond involving Arg105, Gln135 and Thr143 stabilizes the C-terminal α3 helix. The coordinates of 51Z2 have been deposited (PDB: 2M3L).</p

Comparison of HPV 51 and HPV 16 prevalence.

<p>Meta-analysis of prevalence of HPV 51 (blue) and HPV 16 (red) in asymptomatic epithelia (AE; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone.0062584-Bruni1" target="_blank">[75]</a>) and in (pre-)cancerous stages low-grade squamous intraepithelial lesions (LSIL; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone.0062584-Clifford1" target="_blank">[76]</a>), high-grade squamous intraepithelial lesions (HSIL; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone.0062584-Smith1" target="_blank">[4]</a>) and squamous cell carcinoma (SCC; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone.0062584-Smith1" target="_blank">[4]</a>). While the fraction of HPV 16 increases with severity of neoplasia, the HPV 51 fraction decreases after the LSIL stage.</p

Superimposition of 51Z2 to other E6 structures.

<p>Superimposition (details: see SI) of the 51Z2 closest-to mean structure (folded part, residues 80–140, blue) onto the corresponding regions of HPV 16 E6 (red, PDB ID 2LJZ, r.m.s.d. 2.27 Å) and BPV E6 in complex with the LD1 motif of paxillin (gray, PDB ID 3PY7, paxillin omitted for clarity, r.m.s.d. 1.61 Å). The overall topology is conserved. Notably, the β4 and β5 strands and their connecting loop of 51Z2 and BPV position similar, while for HPV 16 E6, the corresponding region orients differently (upper right corner; encircled and highlighted).</p

Resonance assignment of 51Z2.

<p>[¹H,¹⁵N]-HSQC of 1.1 mM 51Z2 with indicated assignments and residue numbering according to full-length E6 sequence. Side chain signals are labeled with SC, and pairs of SC signals are linked by horizontal lines. Two starred resonances: folded signals probably representing arginine-side chains that could not unambiguously be assigned. Experimental details for this spectrum can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062584#pone.0062584.s007" target="_blank">Table S3</a>. The assignment has been deposited in the BMRB (Accession number: 18967).</p

Structure and Biomedical Applications of Amyloid Oligomer Nanoparticles

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimer’s and Parkinson’s. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging