Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization

Figure 1. Human Respiratory Syncytial Virus (hRSV) phosphoprotein P and nucleoprotein N. (A) Domain organization of the RSV P protein (NTD; N-terminal domain, OD; oligomerization domain, CTD; C-terminal domain, itself divided into an α-helix-rich domain Cα and a disordered C-terminal tail, Ctail) and amino acid sequence of C-terminal P peptides. Phosphorylated serines are marked by *. (B) Two views of the X-ray crystal structure of an RSV N-RNA ring containing 10 N protomers (PDB 2wj8) in cartoon representation. The 7-mer RNA molecules are in magenta. One protomer is highlighted in color: N-terminal domain (NNTD) in yellow, C-terminal domain (NCTD) in red, N-terminal arm in green, C-terminal arm in blue. (C) Cartoon representation of the X-ray crystal structure of RSV NNTD (yellow) in complex with the C-terminal P2 peptide (cyan sticks) (PDB 4uc9). (D) Structure of M76 that inhibits the RSV NNTD-PCTD interaction.

Figure 2. Binding of RSV pP11 peptide to RSV NNTD protein followed by NMR. (A) Selected region of superimposed 1H-15N HSQC spectra of 50 µM 15N-labeled NNTD, in 20 mM MES pH 6.5, 250 mM NaCl buffer, recorded during a titration experiment with the pP11 peptide. The magnetic field was 14.1 T, and the temperature 20 °C. The color code corresponds to the pP11:NNTD molar ratio as indicated below the spectrum. (B) Bar diagram of combined amide chemical shift perturbations (ΔδHN) measured for NNTD in the presence of 4.2 molar equivalents of pP11. Regions with significant CSPs are highlighted with a colored background: C-terminus of the αN1 helix (blue), β-hairpin (green), center of the αI2 helix (magenta), η1 loop (cyan), H151-loop (yellow). (C) Mapping of CSPs onto the X-ray structure of NNTD (PDB 4ucc): amide nitrogen atoms are represented as red spheres for residues with |ΔδHN| > mean + 2 SD, and orange spheres for |ΔδHN| > mean + SD. The color code is the same as in (B). The same view in surface representation highlights the PCTD binding pocket, indicated by a white arrow.

Figure 3. Binding of F11 to RSV NNTD and inhibition by M76 assayed by fluorescence polarization. The buffer was MES pH 6.5, with 250 mM NaCl or without salt. The concentration of the fluorescent probe was 200 nM. (A) Binding of NNTD to F11 in buffer with salt: ΔFP = FP − FPmin (the FP of free F11 was FPmin = 30 mFP) was plotted as a function of the NNTD:F11 molar ratio. Error bars represent the difference between duplicate experiments. Data were fitted with a single site binding model (straight line) using Kd and ΔFPmax as parameters. R2 indicates the quality of the fit. (B) Binding of NNTD to F11 in salt-free buffer assayed by ΔFP. The area in the dotted contour box is expanded on the right side. Data were fitted with a single binding site model and an additional linear contribution. (C) Inhibition of NNTD (5 µm) binding to F11 by the M76 Molecule in salt-free Buffer. FP data were fitted with a Hill Equation. IC50 and R2 values are indicated. The dashed line indicates the mean FP value of free F11.

Figure 4. Binding of Fn peptides to RSV N-RNA complex and inhibition by M76 assayed by fluorescence polarization. The buffer was Tris pH 8.0 without salt. The concentration of fluorescent probe was 200 nM. (A) Binding of N-RNA to F11 was assayed by ΔFP as a function of the N:F11 molar ratio. Data were fitted with a single site binding model. (B) Inhibition of N-RNA (10 µM) binding to F11 by M76: The left hand panel represents the experimental FP data, and the right hand panel the percentage of inhibition, calculated assuming FPmax = 120 mFP and FPmin = 40 mFP. (C) Influence of solvent addition on ΔFP measurements for the F7E peptide in the presence of N-RNA.

Figure 5. Binding of FTU-labeled RSV P peptides (Fn) to RSV N-RNA and inhibition by unlabeled peptides assayed by FP. Data were acquired after 30 min incubation. Samples were in Tris pH 8 buffer without salt. The concentration of fluorescent probes was 200 nM. (A) Binding of N-RNA to Fn peptides was assayed by ΔFP as a function of the N:peptide molar ratio for peptides of various lengths. FP of free fluorescent peptide was FPmin ~30–40 mFP. Error bars represent the standard deviation of triplicate measurements. Curves were fitted with a single binding site model. (B) ΔFP binding curves for 11-mer fluorescent peptides (wild-type F11, Ser237Glu mutant F11SE and double Ser232Glu/Ser237Glu mutant F11EE) with N-RNA. Data were fitted with a single binding site model (straight lines). For F11SE a second calculated curve in broken line corresponds to a model with an additional linear component. (C) Inhibition curves of N-RNA (10 µM) binding to F11 by the unlabeled P7E, P9E and P11EE peptides. Data were fitted with a Hill equation. The dashed line indicates the mean FP value of free F11. (D) Inhibition curves of N-RNA (10 µM) binding to Fn peptides by unlabeled P peptides containing the same amino…

Figure 6. Binding of fluorescent molecules to RSV N-RNA and inhibition by M76 assayed by fluorescence polarization. Measurements were carried out in Tris pH 8, Brij-35 buffer without salt. The concentration of fluorescent probes was 200 nM. (A) Chemical structure and name of fluorescent molecules. For fluorescein 1, the open form is shown. Fluorescein methyl ester 2; fluorescein ethyl ester 3; 4,4′ difluoro BODIPY FL 4; 4,4′ dicyano BODIPY FL 5. (B) ΔFP binding curves of fluorescent molecules as a function of the N:fluorescent molecule molar ratio. Fits were performed assuming FPmax = 500 mFP and FPmin = 0 mFP. (C) Inhibition of fluorescent molecule binding to N-RNA (20 µM) by M76.

Figure 7. Analysis of fluorescein binding to RSV NNTD by NMR and by in silico docking. (A) Selected region of superimposed 1H-15N HSQC spectra of 15N-labeled RSV NNTD obtained during titration by fluorescein. NMR data were acquired at a magnetic field of 14.1 T, a temperature of 20 °C, and with 50 µM 15N-NNTD concentration. The color code corresponds to the fluorescein:NNTD molar ratio, as indicated on top. (B) Bar diagram of combined 1H and 15N amide chemical shift perturbations (ΔδHN) measured in the presence of 2 molar equivalents of fluorescein. (C) Mapping of CSPs onto the X-ray structure of NNTD (PDB 4ucc). Amide nitrogens are represented as red and orange spheres for residues with |ΔδHN| > mean + 2 SD and |ΔδHN| > mean + SD, respectively. Regions with significant CSPs are labeled with the same color code on the structure and on the diagram in (B). (D) Two docking poses of fluorescein binding to NNTD: the first pose was obtained with MOE (green) and the second with Smina (blue). (E) X-ray crystal structure of the NNTD-M76 complex (PDB 4ucc).

Figure 8. Binding of dicyano BODIPY FL-labeled C-terminal RSV P peptides to RSV N-RNA and inhibition assayed by FP. Samples were in Tris pH 8 buffer, with or without salt. The concentration of fluorescent peptides was 200 nM. Error bars represent the standard deviation of measurements in triplicate. Fitted curves are in straight lines. (A) ΔFP binding curves of fluorescent BCNn peptides of various lengths to N-RNA in salt-free buffer (FPmin ~ 10 mFP). Data were fitted with a single binding site model. (B) ΔFP binding curves measured in buffer with salt (100 mM NaCl). (C) Inhibition of N-RNA (1 µM) binding to B10EE peptide by unlabeled P10EE peptide and by M76. Data were fitted with a Hill equation. (D) Inhibition of N-RNA (1 µM) binding to two BCNn peptides (BCN10EE and BCN11EE) by unlabeled P peptides with equivalent amino acid sequences (P10EE and pP11). (E) Inhibition by M76 of N-RNA (2 µM) binding to fluorescent peptides BCN5E, BCN6E, BCN8E, and BCN10EE.

Figure 9. Structural model of the RSV NNTD-P11 complex. (A,B) Structures were generated with Haddock software [65], based on the crystal structure of the NNTD-P2 complex (PDB 4uc9) for NNTD and on a P11 model generated with PEP-FOLD3 [64]. 10 models were superimposed. The docked P11 peptides are in yellow cartoon. (A) Two views of NNTD rotated by 90° show the electrostatic surface potential calculated with Delphi software [66], with a scale ranging from −10 (red) to +10 kcal.mol−1 (blue). (B) The backbone of NNTD is shown in cartoon representation. (C) The P11 starting structure used for Haddock docking, in magenta, was aligned with the docked structures represented in other colors. The view is rotated, as compared to (A,B). (D) 20 P11 models generated with PEP-FOLD3 were structurally aligned along the three C-terminal residues Glu239-Phe241. The model used for Haddock docking is in magenta, and the other models in colors unrelated to those used in (C).

Scheme 1. Synthesis of dicyano BODIPY FL 5. (a) BF3.OEt2, TMSCN, DCM, 2 h; (b) THF/H2O/HCl conc, 12 h.