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. 2008 Jan;82(1):173-83.
doi: 10.1128/JVI.01788-07. Epub 2007 Oct 17.

Crystal structure of the NS3 protease-helicase from dengue virus

Dahai Luo  1 , Ting Xu, Cornelia Hunke, Gerhard Grüber, Subhash G Vasudevan, Julien Lescar
Affiliations

Crystal structure of the NS3 protease-helicase from dengue virus

Dahai Luo et al. J Virol. 2008 Jan.

Abstract

Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 A. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain.

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Figures

FIG. 1.
FIG. 1.
Overall structure of the scNS2B18NS3 protein from Den4. (A) Diagram of flavivirus polyprotein organization and the scNS2B18NS3 protein construct used in this work. Proteolytic sites by proteases from the host cell and by NS2B-NS3 are indicated with light and dark blue triangles, respectively. The three predicted membrane-associated regions within the NS2B proteins are represented as filled boxes. A central fragment spanning residues 49 to 66 of the NS2B protein was linked to the full-length NS3 protein through a Gly4-Ser-Gly4 linker. Evolutionarily conserved residues for NS3 enzymatic activities are indicated. (B) Ribbon representation of the scNS2B18NS3 structure. Secondary structure elements are colored in cyan (α-helix) and magenta (β-strand). The three subdomains of NS3hel are numbered. NS2B18, which forms a β-strand, is red. The region linking the protease and helicase (residues 169 to 179) is green. Key residues for NS3 enzymatic activities are shown as sticks and labeled. N-terminal residues are also labeled. A close-up view of the interface between the helicase and protease domains is also shown. (C) View of the electrostatic surface of scNS2B18NS3 with the molecule in the same orientation as above (B) (in the right panel, the molecule is rotated by 180° around a vertical axis). Positive potentials are blue, and negative potentials are red. (D) Side-by-side comparison of scNS2B18NS3 from Den4 and scNS3-NS4A from HCV (48), with their helicase domains oriented similarly. The positions of the protease domain relative to the helicase domain in the two structures are clearly different. The cofactors (NS2B18 and NS4A) are red, and the interdomain linkers are green. The N- and C-terminal residues from the two proteins are labeled.
FIG. 1.
FIG. 1.
Overall structure of the scNS2B18NS3 protein from Den4. (A) Diagram of flavivirus polyprotein organization and the scNS2B18NS3 protein construct used in this work. Proteolytic sites by proteases from the host cell and by NS2B-NS3 are indicated with light and dark blue triangles, respectively. The three predicted membrane-associated regions within the NS2B proteins are represented as filled boxes. A central fragment spanning residues 49 to 66 of the NS2B protein was linked to the full-length NS3 protein through a Gly4-Ser-Gly4 linker. Evolutionarily conserved residues for NS3 enzymatic activities are indicated. (B) Ribbon representation of the scNS2B18NS3 structure. Secondary structure elements are colored in cyan (α-helix) and magenta (β-strand). The three subdomains of NS3hel are numbered. NS2B18, which forms a β-strand, is red. The region linking the protease and helicase (residues 169 to 179) is green. Key residues for NS3 enzymatic activities are shown as sticks and labeled. N-terminal residues are also labeled. A close-up view of the interface between the helicase and protease domains is also shown. (C) View of the electrostatic surface of scNS2B18NS3 with the molecule in the same orientation as above (B) (in the right panel, the molecule is rotated by 180° around a vertical axis). Positive potentials are blue, and negative potentials are red. (D) Side-by-side comparison of scNS2B18NS3 from Den4 and scNS3-NS4A from HCV (48), with their helicase domains oriented similarly. The positions of the protease domain relative to the helicase domain in the two structures are clearly different. The cofactors (NS2B18 and NS4A) are red, and the interdomain linkers are green. The N- and C-terminal residues from the two proteins are labeled.
FIG. 2.
FIG. 2.
Comparison of protease domains from the Flaviviridae. (A) Ribbon representations of Den2 NS2B40NS3 (PDB accession number 2FOM), Den4 scNS2B18NS3 (this work) (PDB accession number 2VBC), WNV NS2B40NS3 (PDB accession number 2FP7), and HCV NS3-NS4A (PDB accession number 1CU1) protease domains. The colors used for the protease domains and their cofactors are cyan and red, respectively. The bound WNV NS3 protease inhibitor (Bz-Nle-Lys-Arg-Arg-H) is shown as yellow sticks. Two β-hairpins (B2B-C2 and E2b-F2) at the C-terminal NS3 protease domain and hairpin (β2 to β3) from the NS2B cofactor are labeled. The side chain of conserved Phe-116, Ile-123, and Val-162 are labeled and presented as purple sticks. The backbone positions of the three catalytic residues (His-51, Asp-75, and Ser-135 in Den4) are orange. (B) Superimposition of the Cα traces of Den4 scNS2B18NS3 (blue), Den2 NS2B40NS3pro (yellow), and WNV NS2B40NS3 (red) (12). NS2B cofactors were removed for clarity. The relative position of the catalytic triad is strictly conserved (green sticks). The main deviation is in the C-terminal region of the protease domain, where two β-hairpins (B2B-C2 and E2b-F2) adopt an open conformation in Den4 compared to the closed conformation observed in Den2.
FIG. 3.
FIG. 3.
The helicase domain. (A) Cα traces for Den4 scNS2B18NS3 (in blue, with the NS2B18 cofactor in red), with Den2 NS3hel in green (PDB accession number 2BMF) (45). The P loop and the loop connecting Arg-460 to Gln-471 in subdomain 2, which are involved in contacts with the protease domain, adopt different conformations. (B) Superposition with YFV NS3hel (in yellow) (PDB accession number 1YMF) (44). A large difference occurs within subdomain 2: residues between Glu-396 and Lys-405 of the YFV helicase are disordered, while the equivalent residues in scNS2B18NS3 fold into an α-helix. The ADP moiety, which was bound to the YFV helicase, is shown as sticks.
FIG. 4.
FIG. 4.
Fluorescence correlation spectroscopy of scNS2B18NS3 and the fluorescent nucleotide analogues in the presence of 2 mM Mg2+. Concentration-dependent binding of scNS2B18NS3 (▪) and NS3hel (□) to ATP ATTO-647N (A) and Mg-ADP ATTO-647N linking to scNS2B18NS3 (▴) and NS3hel (▵) (B) are shown. The percentage of bound nucleotides was analyzed using a two-component binding scheme, [E] + [S] ↔ [ES]. Best fits yielding the binding constants are represented as continuous lines (see the text).
FIG. 5.
FIG. 5.
Interdomain linker between the protease and the helicase domains. (A) Sequence alignment of dengue virus serotypes and WNV scNS2B18NS3 in the linker region (in a rectangle box). Conserved residues are shaded. (B) 2Fo-Fc electron density map for the linker region between the protease and helicase domains. The map is contoured at 1σ for residues 169 to 179, which are shown as sticks and labeled.
FIG. 6.
FIG. 6.
Agreement between the envelope derived from SAXS and the X-ray structure for scNS2B18NS3. (A) Superposition of the low-resolution structure (blue) of scNS2B18NS3 derived from solution X-ray scattering with the crystallographic model (ribbon diagram) (yellow). (B) Experimental SAXS curve from scNS2B18NS3 (1) and scattering from a typical ab initio model of scNS2B18NS3 (2) (computed by use of the program GASBOR) (38). (C) The distance distribution function of scNS2B18NS3 was computed from the experimental data by the program GNOM.
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