Figure 1.
Oligomeric state of recombinant N protein.
(A) Chromatogram of N run on an S200 size exclusion column. The grey line shows the absorbance at 280 nm, and the green line the absorbance at 260 nm. The inset shows a 12.5% SDS-PAGE gel of peaks 1 (N1) and 2 (N2). (B) A 12.5% SDS-PAGE gel showing the presence of dimers, trimers and tetramers after cross-linking fraction N2 with 0.05% glutaraldehyde for one hour at room temperature.
Table 1.
Data collection and refinement statistics.
Figure 2.
Hexamers formed by N in the crystal.
(A) The three N monomers in the asymmetric unit are shown in pink, cyan and green and labeled α, β and γ, respectively. (B) Subunit packing in the crystal layer corresponding to the crystallographic [a,b] plane. The six copies of subunit α that surround the crystallographic 6-fold symmetry axis (black dot) form one hexamer (labeled I), and the three βγ dimers that surround the crystallographic 3-fold symmetry axis (grey dot) form a second hexamer (labeled II). (C) The dimensions of the hexameric ring are labeled. Hexamers I and II face in opposite directions and are offset by approximately 10 Å in the direction of the c axis.
Figure 3.
(A) Sequence of the RVFV N protein with secondary structure elements indicated above. The colors correspond to those used to color the different sub-domains in the crystal structure of N shown in panels B and C. (B) Ribbon representation of the crystal structure of the RVFV N protein showing that the N terminus forms an arm (red) that extends from the globular core domain (brown and green). The C terminus, which is not involved in RNA binding, is shown in blue. The α-helices are labeled. (C) View of the RVFV N protein in surface representation. The orientation and color code are the same as in panel B.
Figure 4.
Interaction between adjacent N subunits in the hexamer.
(A) Overview of how the N-terminal arm of one subunit, shown in orange mesh, fits into the hydrophobic pocket in the surface of the adjacent subunit, shown as grey surface with the hydrophobic pocket highlighted in green. (B) Magnified view of the interaction. The N-terminal arm is shown in ball-and-stick representation and the surface of the hydrophobic pocket is shown transparent to reveal details of the hydrophobic interactions. (C) Amino acid sequence of an RVFV N polypeptide, showing above the secondary structure elements derived from the crystal structure. Below the sequence, residues are labeled that are involved in inter-subunit interactions in the hexamer. The yellow dots indicate residues of the arm that interact with residues in the oligomerization groove. The colored bars indicate the character of the residues in the oligomerization groove: green, hydrophobic; blue, positively charged; red, negatively charged.
Figure 5.
Electrostatic surface potential of the N hexamer.
Mapping of the electrostatic surface potential, from −10 kT in red to+10 kT in blue, onto the surface of hexamer I formed by native N protein reveals a patch of positive charges in the inner part of the ring, which likely accommodates the vRNA. Key residues in the RNA binding site are labeled on the electrostatic surface of a single monomer.
Figure 6.
Electron microscopy of N-RNA complexes.
(A) Representative electron micrograph of the N1 fraction in negative stain, revealing ring-shaped particles of different sizes. Scale bar is 100 nm. (B) Representative class averages of N-RNA rings. (C) 3D reconstruction of a hexameric N-RNA complex obtained with cryo-negatively stained samples. (D) Docking of the crystal structure of hexamer I formed by native N into the EM density map of the hexameric N-RNA complex.
Figure 7.
Gallery of crystal structures of N proteins from different negative strand RNA viruses.
The structures shown are those of the N proteins from the rabies virus (RV; PDB code: 2GTT), the vesicular stomatitis virus (VSV; PDB code: 2GIC), the Rift Valley fever virus (RVFV; this work), the respiratory syncytial virus (RSV; PDB code: 2WJ8), and the influenza virus (InfV; PDB code: 2IQH). The model fro the influenza NP ring is derived from an EM reconstruction into which the crystal structure of the monomer was modeled (PDB code: 2WFS). The top three rows show different views of the ring structures in surface representation, in which each subunit is shown in a different color. The fourth row shows two views of the monomers in ribbon representation. The arrows indicate the cavity that binds the vRNA.