Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Abstract

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an "induced fit" mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

Figure 1.

Structure and organization of the flaviviral NS2B-NS3 proteases. (A) Organization of the polyprotein precursor, showing cleavage points by the NS2B-NS3 protease (green arrows) and host cell proteases (black arrows), with detail of the NS2B and NS3 sequences, and (bottom) the fusion construct used in the present study. (B) Ribbon representations of (left) aprotinin-bound WNV NS2B-NS3pro and (right) aprotinin-free WNV NS2B-NS3pro with secondary structural elements and N and C termini indicated. NS3 is shown in green (N-terminal lobe), gray (C-terminal lobe), and orange (the linker between the two lobes); NS2B is in purple; aprotinin is in yellow. The catalytic triad is shown as black balls. (C) NS3 sequences of the flaviviruses: West Nile virus (WNV), Japanese Encephalitis virus (JEV), Kunjin virus (KV), Dengue virus serotypes 1–4 (DV1–4), Yellow Fever virus (YFV); and the hepacivirus, Hepatitis C (HCV). Red highlight indicates identity, red letters homology. Secondary structure elements above the sequences are for WNV NS2B-NS3pro; those below are for HCV NS3pro-NS4A (PDB entry 1JXP). Gray rectangles highlight regions where the folds of WNV NS2B-NS3pro and HCV NS3pro-NS4A differ. The seven-residue insertion unique to the flaviviruses is boxed in green. The structural elements of cofactor-free DV NS3pro (see Fig. 5) are identical to those of HCV NS3pro-NS4A, but the majority of its β-barrel-forming strands have incomplete H-bonding and are not classified as true sheets. (D) Sequences of flaviviral NS2B and HCV NS4A. Boxes indicate minimal cofactor segments required for activation of the NS3 protease in vitro. TM1–TM4 are predicted transmembrane regions. Hydrophobic and aromatic residues are in green, polar in black, acidic in red, and basic in blue. Secondary structure elements are for the WNV protease–aprotinin complex; the alternate elements (α1 and β1′) found in inhibitor-free WNV and DV NS2B-NS3pro are drawn with dotted lines.

Figure 2.

Structure of the NS2B-NS3–inhibitor interface and evidence for induced fit. (A) Stereo view of WNV NS2B-NS3pro surface with selected aprotinin residues. The green and blue sticks are two parts of aprotinin (residues ¹³PCKARII¹⁹ and ³⁵GGCR³⁹) that interact with the protease. The surface is colored by electrostatic potential (negative, red; positive, blue). The magenta ribbons show the invading β2–β3 hairpin of NS2B contributing to the active site. Selected residues of the protease are shown as sticks. (B,C) Comparison of the oxyanion hole conformations in the WNV NS2B-NS3pro complexed with (B) aprotinin and (C) a peptidic inhibitor (Erbel et al. 2006; PDB code 2FP7). Selected residues of the protease (yellow) and inhibitors (green) are shown. In B, aprotinin induces the active conformation of the oxyanion hole, which is occupied by the P1 C=O. In C, the oxyanion hole is disrupted owing to a flip of the peptide bond at Gly133–Thr132, which creates a 3₁₀ helical conformation (the C=O of Thr132 is marked with an arrow in B and C). The P1 C=O and P1′ side chain of aprotinin (shown as black stick in C) would clash with Thr132 in this conformation. Selected hydrogen bonds are shown with dashed lines.

Figure 3.

Substrate specificity at the S1′ pocket. Close-up of the surfaces of aprotinin-bound (A) WNV NS2B-NS3pro and (B) bovine trypsin, showing the pronounced pocket in the WNV protease that is absent in trypsin (where a disulfide bridge occludes the pocket). Ala of aprotinin has been replaced in silico by Thr, a consensus residue for flaviviral substrates. Potential H-bonds to NS3 residues Ala36 and Thr132 are shown as dashed lines in A, and the oxyanion hole (blue) occupied by the P1 C=O is evident at the center of both figures. WNV NS2B-NS3pro actually prefers glycine, perhaps owing to the additional H-bond to Thr132, which restrains the P1–P1′ dihedral angles. (C) Stereo stick comparison of A and B with WNV complex in yellow and trypsin in green. Selected H-bonds are shown as dashed lines. (D) WNV NS2B-NS3pro cleavage sequences, showing consensus at the P2, P1, and P1′ positions.

Figure 4.

Two conformations of the NS2B cofactor. (A) Superposition of WNV NS2B-NS3 proteases: substrate-free (green and blue) and aprotinin-bound (gray and red). The secondary elements of NS2B unique to the substrate-free (α1 and β1′) and the substrate-bound (β2 and β3) structures, as well as the alternative C termini are indicated. The point of departure (Trp62) for the two NS2B elements is labeled and shown as a stick model. The elements β2′ and β3′ in the substrate-free structure are stabilized by crystal contacts (see Supplemental Fig. 3). Active site residues are shown as black circles. (B) Superposition of substrate-free DV (gray and red) and WNV (green and blue) NS2B-NS3pro. The collapse of the E2B–F2B loop observed in DV is indicated by the arrow.

Figure 5.

Stereo comparisons of flaviviral and HCV protease domains. (A) HCV NS3pro-NS4A protease (PDB entry 1JXP). NS4A is in purple; NS3 is in yellow, except for regions analogous to the mobile regions in flaviviruses (in blue). Hydrophobic side chains on the NS3 N-terminal helix implicated in membrane attachment are in green. (B) Cα superposition of HCV NS3pro-NS4A (blue/magenta) and cofactor-free DV NS3pro (green; PDB entry 1DF9). The RMSD between Cα atoms is 1.44 Å for 157 residues. The seven-residue insertion unique to flaviviruses (residues 117–124 in WNV) is highlighted in red. (C,D) Cofactor-free DV NS3pro (C) and aprotinin-bound WNV NS2B-NS3pro (D) colored to illustrate conformational differences. Regions of NS3 that are structurally similar are in yellow; those that acquire alternate conformations upon cofactor binding are in blue, except for residues 26–32 and 117–123, which are shown in red to highlight their major relocations. Cofactor binding leads to a separation of the C-terminal barrel into an open “lower barrel” (LB) and a closed “upper barrel” (UB). Catalytic residues, selected side chains that may be important for the conformational changes (Pro/Val106 and Pro113), and the hydrophobic hairpin at the A1–B1 turn of NS3 exposed by NS2B binding are shown as green sticks. NS2B is in purple. Selected secondary structure elements are labeled. β-strands of DV NS3 that do not form regular β-sheet H-bonds are shown as loops.