PMC

Structure of the P7 core (P7ΔC). (a) Topological diagram for P7ΔC generated using the PDBsum server ; ; . Secondary structure assignments are from PROMOTIF v3.0 . (b) Side-by-side stereo-view of P7ΔC. Key structural elements are labeled and the two 3₁₀ helices are colored cyan.

Slow dynamics of the P7ΔC backbone. (a) R_ex values obtained from the model-free analysis of the relaxation data at 600 MHz. R_ex > 10 s⁻¹ (red), 5–10 s⁻¹ (green) and < 5 s⁻¹ (blue). (b) The residues that display R_ex values are mapped onto the structure of P7ΔC, the largest R_ex values are seen in the hydrophobic core and at the dimer interface.

P7/RNA interactions. (a) Surface plot of P7ΔC colored by electrostatic potential. Residues on P7ΔC that show chemical shift changes > 0.04 ppm in the presence of RNA (P7ΔC:Oligo2 ratio 1:2) are labeled. (b) Residues that undergo large chemical shift changes in P7 in the presence of RNA are shown. Red, blue and green spheres represent acidic, basic and hydrophobic residues respectively. The dynamic N- and C-terminal tail conformations are represented schematically.

The flexible C-terminal tail of P7 interacts minimally with the protein core. (a) Overlay of ¹⁵N,¹H TROSY spectra (800 MHz) of ¹⁵N,REDPRO-²H-labeled P7fl (black) and P7ΔC (red). The Ala98 resonance is aliased. Also shown are the intense resonances corresponding to the C-terminal tail residues in a ¹⁵N,¹H HSQC spectrum (600 MHz, cyan) of fully-protonated P7fl. The broad resonances corresponding to the protein core are not seen when the spectrum is suitably contoured. The C-terminal tail resonances were independently assigned in the context of P7fl. Peaks have been shifted to compensate for the ¹J_NH couplings in both dimensions. The boxed region is expanded in (b). (b) Resonance assignments of the flexible C-terminal tail resonances in fully-protonated full-length P7 (P7fl).

Interactions of P7ΔC with RNA. (a) Expanded region of a ¹⁵N,¹H HSQC spectrum (acquired at 600 MHz) of ¹⁵N,²H-REDPRO-labeled P7ΔC with varying amounts of Oligo2 (see text). Residues that undergo large chemical shift changes (scaled shift changes calculated using Equation 3 for a P7ΔC:RNA ratio of 1:2) are labeled - 0.04 ppm < Δδ < 0.07 ppm are labeled in green, 0.07 ppm ≤ Δδ < 0.10 ppm are labeled in blue and Δδ > 0.10 ppm are labeled in red. (b) Scaled chemical shift changes plotted against residue number for a P7ΔC:RNA ratio of 1:2. Key residues with large shift changes ( > 0.04 ppm) are labeled using the same color scheme as in (a). (c) Two specific surfaces display large chemical shift changes in the presence of RNA – key residues are labeled and the coloring scheme used is as in (a).

Interactions of the C-terminal tail in full-length P7 with RNA. (a) Expanded region of a ¹⁵N,¹H HSQC spectrum (acquired at 600 MHz) of fully-protonated ¹⁵N-labeled P7fl in the presence of varying amounts of Oligo1 (see text). Substantial shift changes were are also seen for Gly151 and Gly162 (not shown in the expansion). The spectra are plotted at a suitable contour level to allow clear visualization of the sharp peaks corresponding to the C-terminal tail. (b) Scaled chemical shift changes occurring for tail-resonances of P7fl in the presence of RNA at a 1:1 molar ratio. Residues that are broadened beyond the observation threshold are labeled in red. Other key residues that show substantial chemical shift changes are also labeled.

The C-terminal residues in full-length P7 are highly disordered. (a) Steady-state ¹⁵N-{¹H} NOE data at 600MHz for the 41 C-terminal tail residues in fully protonated full-length P7. (b) The difference in the deviation of the C^α and C^β chemical shifts from the corresponding random coil values for the 41 C-terminal residues in fully-protonated full-length P7 (P7fl). A string of positive values at the N-terminal end of this tail region indicates helical propensity. (c) NOE correlation patterns for the 41 C-terminal residues in full-length P7. NOEs to the backbone amide H^N are classified as strong, medium and weak represented by the thickness of the horizontal bars. Also shown are the ³J(H^NH^α) values - < 6.5 Hz (filled circles), 6.5–8.5 Hz (open squares) and > 8.5 Hz (filled triangles).

Sequence conservation in cystoviral P7 proteins. (a) Sequence alignment for the P7 proteins for the ϕ6, ϕ8, ϕ12 and ϕ13 cystoviruses are shown. The residues that comprise the dimer interface are boxed. The regions of definite secondary structure are indicated – α-helices (thick red line), 3₁₀-helices (thick cyan line), β-strand (green arrow). The dotted black line indicates the residues that have no electron density in the crystal structure of P7ΔC. The flexible C-terminal tail is indicated by the dotted magenta line. (b) Conserved surface residues in P7. Highly conserved residues are colored blue and strictly conserved residues are colored red. (c) Residues that show R_ex values > 4.5 s⁻¹ for P7ΔC are shaded green. Residues for which ¹H^N,¹⁵N assignments could not be obtained in the TROSY spectra of P7ΔC are shaded gold.

Fast dynamics of the P7ΔC backbone. (a) S² values were obtained by a model-free analysis utilizing backbone R₁, R₂ and steady-state ¹⁵N-{¹H} NOE data at 600MHz. Data were fitted to an anisotropic model using the NH unit vectors generated utilizing the crystal coordinates. The microdynamic parameters of the flexible N-terminus of P7ΔC, for which no crystal coordinates were available, were obtained by fitting to an isotropic model with the same effective correlation time as the anisotropic model. S² values for the anisotropic and isotropic fits are represented by filled and open circles respectively. The regions of definite secondary structures are shown schematically – helices (α – dark, 3₁₀ – light) by springs and β-strands by arrows. Phe74 and Val100 that have an unusually low S² values are labeled. (b) Reduced spectral density values near the ¹H Larmor frequency – J(0.87ω_H). Residues with increased J(0.87ω_H) values due to extensive sub-nanosecond timescale dynamics are labeled.

Structure of the P7ΔC biological unit. (a) P7ΔC forms a symmetric homodimer in solution. The monomers are colored blue and red. (b) The dimerization surface of P7ΔC is composed of mostly hydrophobic residues. Key residues forming the dimerization surface are labeled. Hydrophobic residues are colored green, the basic Arg77 and the acidic Asp69 are colored blue and red respectively. (c) The backbone amide H^N and O atoms of Met49 and Ala80 are involved in intermolecular hydrogen bonds, as are the sidechains of Arg77 and Asp69. The two monomers are represented by ribbons colored red and blue with key residues shown in stick representation colored orange and cyan. Hydrogen bonds are shown by green dotted lines. (d) Gel filtration data (Pharmacia Superdex 200 HR 10/30) showing elution volumes of molecular weight markers (black) and P7fl (D69R). The Asp to Arg mutation disrupts dimer formation in P7.