Figure 1.
Schematic representation of HPV-16 E7 oncoprotein and the E7N Intrinsically disordered domain.
NMR structure of the dimeric HPV-45 E7C domain (PDB ID: 2F8B) with each monomer represented in blue and grey respectively and the structural Zn2+ atoms shown as red spheres. The E7N domain from a single E7 monomer is shown as a ribbon with the aminoacidic sequence for HPV-16 detailed in black. Conserved regions CR1 and CR2 are boxed with targets that bind to each region shown in colored boxes above the sequence. (*) Indicates proteins that bind to both conserved regions. The N-terminal ubiquitination site (Ub) and the E7N linear motifs (DYRK1A, E2F-MIMIC, LxCxE, CKII and acidic PEST region) are underlined. The region covered by the peptide fragments used in this work is shown by colored lines below the sequence. E7 (1–20): blue line; E7 (16–40): orange line; E7 (16–31): red line; E7 (25–40): violet line and E7 (21–29): green line.
Table 1.
Aminoacidic sequences of E7N sub-fragments used in this work.
Figure 2.
Dissection of transient secondary structure elements within E7N.
A) Far-UV CD spectrum of E7 (1–40) and its sub-fragments in 10 mM Tris.Cl pH 7.5 buffer at 20°C. Shown are E7 (1–40): black line; E7 (1–20): blue line; E7 (16–40): orange line; E7 (16–31): red line; E7 (21–29): green line and E7 (25–40): violet line. B–C) Stabilization of pre-existent α-helix structure with 53% TFE for fragments E7 (16–31), E7 (25–40) and E7 (21–29) measured at 20°C in 10 mM Tris.Cl buffer at pH 7.5 (B) and 10 mM sodium formate buffer at pH 5.0 (C). The vertical dotted lines indicate 208 nm and 222 nm wavelengths and the fragment coloring code is as in (A).
Figure 3.
Characterization of α-helical populations within E7N by CD spectroscopy.
A) Far-UV CD spectra of E7 (16–31) in 20 mM Tris.Cl buffer pH 5.0 at 20°C and TFE % (v/v) percentages ranging from 0 to 53%. The arrow indicates the sense of change upon increasing TFE. B) Titration curves for E7 (16–31) following ellipticity at 222 nm as a function of [TFE]/[buffer] molar ratio at pH 7.5 (full circles) and 5.0 (open circles). The lines show fitting of the data to a two-state coil-helix equilibrium model (see Materials and Methods and Table 2). C) Maximum percentage of α-helix content induced by TFE in E7N and the sub-fragments using 20 mM Tris.Cl buffer pH7.5 (white bars) and 20 mM sodium formate buffer pH 5.0 (gray bars). The percentage of residues in α-helix conformation was calculated from data fitting of TFE titrations for each fragment to a two-state coil-helix equilibrium model (Figure S1 and Table 2). The symbol (*) indicates no alpha helix induction. D) pH titration curves for different fragments followed by molar ellipticity at 222 nm in 10 mM citrate-phosphate buffer containing 30% TFE. E7 (1–40): dark circles; E7 (16–31): open circles, and E7 (1–20): open squares. Fitting of the data to Equation 4 are plotted as full lines.
Table 2.
Parameters for α-Helix Stabilization in HPV-16 E7 protein and peptides.
Figure 4.
pH dependence α-helix formation in HPV-16 E7, the E7 (27–98) truncated variant and E7N.
A–C) Far-UV CD spectra of E7 (1–98) (A), E7 (27–98) (B), and E7 (1–40) (C). Spectra were measured at 20°C in 10 mM Tris.Cl buffer pH 7.5 (full line), 10 mM Tris.Cl buffer pH 7.5 with 53% TFE (broken line), 10 mM sodium formate buffer pH 5.0 (dotted line) and 10 mM sodium formate buffer pH 5.0 with 53% TFE (dotted and dashed line).
Figure 5.
Effect of the anionic detergent SDS on secondary structure of E7N and its fragments.
A–B) Far-UV CD spectra of E7 fragments in 10 mM sodium formate buffer pH 3.0 containing 25 mM SDS (A) or 1 mM SDS (B). Fragments are E7 (1–40): black line; E7 (1–20): blue line; E7 (16–40): orange line; E7 (16–31): red line, and E7 (25–40): violet line. The critical micellar concentration (CMC) calculated for SDS in this experimental condition is 5 mM.
Table 3.
Hydrodynamic properties of E7 (1–40), E7 (1–20) and E7 (16–40).
Figure 6.
E7N chemical shifts assignments.
A) Finger-print region of 2D 1H-1H NOESY spectrum of E7 (1–40) in aqueous solution at pH 5.0, showing sequential NOE connectivity among Hα nuclei. For clarity, only NH-Hα (i, i) and NH-Hα (i+1, i) cross peaks are indicated in the figure. B) Selected region of the 1H-13C HMQC spectrum of E7 (1–40) in aqueous solution at pH 5.0. The assignment of 13Cα-1Hα cross-peaks is shown. The inset shows the glycine assignment.
Figure 7.
Determination of transient α-helixes within E7N by chemical shifts analysis.
1Hα and 13Cα, corresponds to chemical shift differences between aqueous and aqueous-TFE solutions for 3 mM E7 (1–40) in 50% TFE-d2 at pH 7.5 (A) and in 12%TFE-d2 at pH 5.0 (B). TFE content differs at both pHs, leading to different magnitude of the chemical shifts differences at pH 7.5 and pH 5.0. Regions with transient α-helical conformation are shaded in blue. The E7 (1–40) aminoacidic sequence is shown in the upper part of the figure with acidic residues shown in red and histidines in blue.
Figure 8.
Polyproline type II propensity within E7N.
A) Far-UV CD spectra of E7 (25–40) in 10 mM buffer Tris.Cl pH 7.5 at 5°C (broken line), 25°C (full line) and 75°C (dotted line). Inset: Difference spectrum between 5°C and 75°C (black line) and between pH 8.0 to pH 3.0 at 30% TFE for the E7 (25–40) fragment (red line; data taken from CD spectra in Figure 2). The arrow shows the positive band at 218 nm characteristic of PII structure. B) Molar ellipticity at 218 nm (Δθ218) obtained from the difference spectrum between 5°C and 75°C for each E7 peptide. White bars: 10 mM buffer Tris.Cl pH 7.5. Dark bars: 10mM buffer sodium formate pH 5.0. C) Plot of the differences between observed and random coil 1JCαHα values vs. the position along the E7N sequence, for aqueous solution pH 7.5 at 4°C (upper panel) and for aqueous solution pH 5.0 at 20°C (lower panel). Regions that populate α-helical and PII conformations in each pH condition are shaded in blue and red, respectively. The E7N amino acid sequence is shown in the upper part of the figure with acidic residues and histidines shown in red and blue respectively.
Figure 9.
Model of E7N conformational ensembles at pH 7.5 and pH 5.0.
In the upper part of the figure the E7N sequence is shown with CR1 and CR2 regions boxed in blue and orange, respectively. The Ubiquitination site (Ub), and the DRYK1A, E2F-MIMIC, LxCxE Rb-binding and CKII/PEST motifs are displayed above the sequence. Phosphoserine residues are indicated with red ovals and regions that stabilize α-helix and PII by punctuated rectangular boxes. The lower part of the figure shows snapshots indicating the particular elements of secondary structure that are populated by the E7N conformational ensemble at pH7.5 and pH5.0 with arrows indicating conformational equilibria and partial population of secondary structure elements. E7N regions show the same color-coding as the upper panel, with the location of helixes and PII structure indicated by boxes and ovals respectively.