Figure 3.

Binding of flock house virus B2 (1–72) to double-stranded RNA. (A) Chemical shift perturbation after addition of a 21-mer short interfering RNA (siRNA) duplex to a ²H-, ¹³C-, ¹⁵N-labelled flock house virus (FHV) B2 dimer. The chemical shifts are monitored in ¹H, ¹⁵N correlation spectra. The spectrum of the free protein is shown in black and the spectrum after addition of a twofold molar excess of siRNA is shown in red. The dimer concentration was 0.25 mM. (B) Titration curves of double-stranded RNA (dsRNA) binding to FHV B2. The fraction of bound protein (f_bound) is plotted as a function of ligand concentration. f_bound is defined by the signal intensities, f_bound=int_bound/(int_bound+int_free), where int_bound and int_free are the peak intensities of the RNA-bound and free NMR signals (supplementary Fig S2 online). The inflection point of the titration curve at equimolar dimer–RNA ratio indicates a 1:1 stoichiometry for the B2 dimer–RNA complex. (C) Left: surface representation of the B2 dimer coloured according to chemical shift changes induced following the addition of siRNA (see (A)). Residues that are affected/not affected by the RNA addition are shown in magenta/white. Residues that could not be analysed unambiguously because of signal overlap are shown in light yellow. The same orientation as in Fig 1A is shown. Middle: the same surface coloured in blue and red according to positive and negative electrostatic potential, respectively. Right: a ribbon representation in the same orientation. Side chains of residues discussed in the text are shown. (D) Proposed interface of the B2 dimer with dsRNA. The B2 dimer is shown rotated by 90° around the y axis compared with (C). Side chains of lysine and arginine residues in the RNA-binding surface are shown. Distances between these residues and the total length of the RNA-binding surface are indicated.