Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications

Abstract

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 A of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B(18)NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by approximately 161 degrees with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn(2+) refined to a resolution of 2.2 A. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu(173) and Pro(174) or replacing Pro(174) with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro(176) to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

FIGURE 1.

Alternative conformations for the flavivirus NS2B₁₈NS3 protein. A, side-by-side view of the two crystallographic conformations of NS2B₁₈NS3 that occur using similar crystallization conditions. Secondary structure elements are colored in cyan (α-helix) and magenta (β-strand). The NS2B₁₈ cofactor that forms a β-strand is colored in red. The region linking the protease and helicase (residues 169–179) is colored in green. N-terminal residues are also labeled. The rotation axis that relates both protease domains is indicated with the corresponding angle. B, docking ADP-Mn²⁺ into NS2B₁₈NS3 in its conformation I (in magenta, PDB code 2VBC). For comparison, NS2B₁₈NS3 in its conformation II is superposed (colored in green). The figure was generated following superposition of the helicase domain of the two crystal structures.

FIGURE 2.

Conformations of the linker between the protease and helicase domains. A, linker region (colored in green) of NS2B₁₈NS3 in conformation II. The electron density map with Fourier coefficients 2|F_o| − |F_c| is shown for the linker region contoured at 0.8σ for residues ¹⁶⁹ERIGEPDYEVD¹⁷⁹. B, linker region of NS2B₁₈NS3 in conformation I (mutant E173GP174). The electron density map is contoured at 1σ for residues ¹⁶⁹ERIGEGPDYEVD¹⁸⁰. C, side-by-side view of the linker regions for the three crystal structures as listed in Table 1. Residues Glu¹⁷⁷ to Phe¹⁸³ are displayed as sticks and labeled.

FIGURE 3.

ATPase/helicase activities of the wild type NS2B₁₈NS3, its mutants P174G and E173GP174, and the NS3 helicase domain protein. A, ATPase activity assay was carried out with 4.8 nm protein in the presence of varying concentrations of ATP (0–0.8 mm). The inorganic phosphate released during catalysis was measured with the Malachite green reagent at 620 nm, and the initial rates were determined (data not shown) as described under “Materials and Methods.” The K_m and k_cat values were calculated using the Lineweaver-Burk plot (see Table 5). Both mutants show around 50% reduction in catalytic efficiency compared with NS2B₁₈NS3 and are comparable with the helicase domain. B, unwinding activity comparisons between mutant and wild type proteins using a nonradioactive assay. The DNA oligonucleotide (D1) was labeled with biotin at its 3′ end, so that it can be separated on a nondenaturing gel and transferred to Hybond-P polyvinylidene difluoride membrane for detection as described under “Materials and Methods.” The unwinding activity assay was carried out three times, and a typical result is shown. Lanes 1, 3, and 5 are samples at zero time point, and lanes 2, 4, and 6 are samples at the end of 30 min of incubation. The intensity of the lower band was detected using QuantityOne software, with the intensity of WT full-length defined as 100%. C, bar graph shows a relative helicase activity comparison (% of WT full-length). The WT helicase domain protein and the Gly insertion mutant (E173GP174) are around 30% active, and P174G is around 60% active.

FIGURE 4.

Expression of wild type and P176G replicon. A, schematic representation of the Rluc-replicon in which the majority of structural gene-encoding regions have been replaced by a puromycin resistance gene (pac), a FMDV2a cleavage site, Renilla luciferase (Rluc) reporter gene, and internal ribosomal entry site. These are flanked by remnants of the C and E proteins, i.e. N-terminal 22 residues of C (C22) and C-terminal 24 residues of E (E24). The construction of DENrepPAC2A-Rluc_NS3P176G involves the following: (i) removal of the KpnI site in NS5 via overlapping extension PCR; (ii) site-directed mutagenesis of NS3 proline 176 to glycine (P176G). This 6.2-kb KpnI-XbaI fragment cloned in pVAS5 was digested accordingly and ligated to XbaI-digested and KpnI partially digested (only at KpnI site of NS2) Rluc replicon, to yield DENrepPAC2A-Rluc_NS3P176G. B, figure shows percent luminescence from the average of two separate experiments carried out in triplicate. Mutant replicons 11 and 22 are two separate clones with the same mutation (P176G). BHK21 cells were electroporated with WT and mutant replicon as described under “Materials and Methods” along with a β-galactosidase plasmid. The luminescence was measured and normalized against β-galactosidase activity. The data were plotted as percent luminescence with respect to the wild type replicon luminescence. C, real time reverse transcription-PCR was performed on cells electroporated as in B with primers for NS1 and actin using conditions described under “Materials and Methods.” The data were plotted as a bar graph with percent change in viral RNA level compared with the wild type replicon RNA level. The results are the mean from two separate experiments carried out in triplicate.

FIGURE 5.

Model for the membrane-bound NS2B-NS3 protein complex. Left panel, proposed association with the membrane of NS2B-NS3 when it adopts conformation I (generated by superposition of the protease domain of NS2B₁₈NS3 in conformation I to the membrane-associated NS2B-NS3 protease). The predicted topology of NS2B protein assumes that the essential cofactor (residues 49–96) is tethered to the membrane via the N- and C-terminal membrane anchoring regions. The membrane-associated NS2B-NS3pro model is generated based on comparisons with the apoprotein structure of DENV2 NS2B₄₇NS3pro (PDB code 2FOM) and with substrate-bound structure of West Nile virus NS2B₄₇NS3pro (PDB codes 2FP7 and 2IJO) (2, 3). The model places an exposed hydrophobic loop (Gly²⁹–Leu³⁰–Phe³¹–Gly³²), labeled as GLFG, next to the membrane that has been experimentally shown to associate with membrane (see supplemental Fig. 4 and supplemental Table 1). Putative membrane association points are indicated as black triangles (GLFG loop, residues 48 and 97 of NS2B). Missing residues in the crystal structures are displayed as dotted lines. The hydrophilic region of NS2B cofactor (NS2B₄₇) is in red. NS3 is in green. Residues from the catalytic triad His⁵¹, Asp⁷⁵, and Ser¹³⁵ are shown as sticks and labeled. The model for the membrane (phosphatidylcholine) was generated using VMD (41). Note that the single strand RNA binding groove within the helicase domain as well as the C terminus of NS3 are facing toward the membrane. Right panel, membrane association of NS2B-NS3 (conformation II). The model was generated by superposition of the protease domain of NS2B₁₈NS3 in its conformation II to the membrane-associated NS2B-NS3pro. In this conformation, the helicase domain projects away from the membrane and is thus more accessible to RNA as well as the NS5 methyltransferase-polymerase. The orientation of the NS3 helicase from Murray Valley encephalitis virus is included for comparison (see text).