Figure 1.
Crystal structure of CSFV Npro.
(a) Ribbon representation of wild type and (b) Stereoview of the C168A Npro. The protease domain is shown in red and the zinc-binding domain in green. Disordered residues (65 to 71 in wild type and 145 to 149 in C168A mutant) are indicated by dashed lines. Secondary structural elements, as well as the N- and C-termini, are labeled. (c) Ribbon diagram of Npro-C168A mutant and its symmetry mate (grey) involved in zinc coordination at the catalytic site. Amino acids involved in the coordination complex (Cys69, His49, terminal carboxylate of Ala168 of one monomer and His74' from the neighboring monomer) are shown in sticks. Zinc atom is shown in magenta. Inset shows the close-up view of the zinc-binding site with the 2Fo-Fc electron density map contoured at 2σ (cyan). The anomalous difference Fourier map (blue) calculated with data collected at the zinc absorption edge (λ = 1.2823) is contoured at 8σ. (d) Sequence alignment of pestivirus Npro. The alignment includes the CSFV strain Alfort/187, Border Disease virus (BDV) strain AV, BVDV-2, Pestivirus giraffe-1, and the Bungowannah virus (GenBank accession numbers X87939.1, ABV54604.1, AAV69983.1, NP_777520.1, and DQ901403.1, respectively). Amino acid numbering corresponds to the CSFV Alfort/187 sequence. The conserved residues are in bold, the cysteine protease dyad is in red and the TRASH sequence motif is in blue. The domains are color coded as in (a). The secondary structure elements are indicated below the sequence. α-helix and β-strands are shown as coil and arrows, respectively.
Table 1.
Data collection, phasing and refinement statistics.
Figure 2.
(a) The N-terminal protease domain of Npro. The catalytic dyad (Cys69 and His49) is shown in sticks. Glu22 originally predicted to be a part of the active site is at a distance of 23 Å from Cys69 and forms a salt bridge (dashed lines) with Arg100. (b) Stereo view of the active site of Npro. The catalytic dyad (Cys69 and His49) flanks the cleavage site Ala168. The distances in Å between the catalytic dyad and the terminal carboxylate in the active site are shown in black dashed lines. The hydrogen bonding distances between Ala168 and the oxyanion hole formed by the amide hydrogens of Gly67, Asp68 and Cys69 are also indicated (magenta dashed lines). (c) Substrate binding site of Npro. The C-terminal β-strand (β8), containing substrate sites P1 through P6, is shown in yellow, along with the surrounding residues forming the substrate binding site in the protease domain (white). The adjoining strand β1 that stabilizes β8 by main-chain hydrogen bonding interactions is also shown and labeled. (d) Zinc-binding domain of Npro. The TRASH motif (Cys112-Cys134-Asp136-Cys138) is shown in stick models on one end of the β-sheet. Zinc is missing in the structure and Cys112 and Cys134 form a disulfide bond, probably due to oxidizing conditions in the crystallization solution.
Figure 3.
Spatial distribution of the residues involved in Npro-mediated proteasomal degradation of IRF3.
The residues that are essential for the Npro-mediated IRF3 degradation (red), along with the conserved residues (yellow, bold letters in Fig. 1) are mapped onto the surface of Npro. The residues localize to two protein surfaces, one in each domain. The protease domain surface cluster (left) includes residues Glu22 and His49, and the zinc-binding domain cluster (right) is formed by the residues in the TRASH motif.