Ns5

FIG. 2. Side by side view of dengue virus NS3 (left) and HCV helicase catalytic domains (right) (Kim et al 1998) displayed in the same orientation. Only the a-carbon atoms traces are shown. Their structurally conserved RecA-like domains I and II are shaded in grey. Their domains III bear no significant structural similarity. The putative binding site of NS5 is indicated.

FIG. 2. Side by side view of dengue virus NS3 (left) and HCV helicase catalytic domains (right) (Kim et al 1998) displayed in the same orientation. Only the a-carbon atoms traces are shown. Their structurally conserved RecA-like domains I and II are shaded in grey. Their domains III bear no significant structural similarity. The putative binding site of NS5 is indicated.

FIG. 3. Comparison of flavivirus helicase catalytic domains. The Ca traces of yellow fever virus and dengue virus helicases were superimposed, giving a r.m.s deviation of 2.3 A for 415 residues.

The NTPase active site

The NTP substrate is primarily bound through ionic interactions through its phosphate moieties whilst the ribose and base bulge out from the NTP binding cavity (Wu et al 2005, Xu et al 2005). As a result, there are few structural constraints for their recognition by the enzyme, an observation consistent with a lack of specificity for the base. This is also consistent with the capacity of NS3 to hydrolyse short oligo-ribonucleotides regardless of the nature of the base moiety (Benarroch et al 2004). We obtained a crystal form (Table 1), with a Mn2+ and a sulfate ion from the crystallization buffer located in the ATP binding pocket. The cation makes hydrogen bonds with the carboxylate group of Glu285 of the DExH motif II and is further coordinated by six water molecules (Fig. 4). A comparison of the orientations of the residues in this cavity with the yellow fever virus helicase bound to ADP (Wu et al 2005) shows that a sulfate ion is located at a position close to the y-phosphate of an ATP substrate. Thus, this structure might resemble a product state that would follow the release of ADP from the NTP binding pocket.

TABLE 1 Refinement statistics (Mn2+ complex)

Resolution range (A)

20.0 - 2.75

Intensity cutoff (F/a [F])

0.

No of reflections: completeness (%)

97.7

Used for refinement

24536

No of non hydrogen atoms

1243

Protein

3140, 3480

missing residuesa

(38, 11)

SO42-

3

Water molecules

229

Mn2+

2

Rfactorb (%)

20.6

Rfreec (%)

26.4

Rms deviations from ideality

Bond lengths (A)

0.0071

Bond angles (°)

1.401

Ramanchandran plot

Residues in most favoured regions (%)

84.0

Residues in additional allowed regions (%)

16.0

a Values are given for molecule 1 and 2, respectively. b Rfactor = Z ||Fob,| - |F„J| / Z |Fob,|.

c Rfree was calculated with 5% of reflections excluded from the whole refinement procedure.

a Values are given for molecule 1 and 2, respectively. b Rfactor = Z ||Fob,| - |F„J| / Z |Fob,|.

c Rfree was calculated with 5% of reflections excluded from the whole refinement procedure.

Nucleic acid binding sites

A tunnel lined with a number of basic residues runs approximately through the centre of the structure. Based on previous structural work on the HCV and PcrA helicases (Yao et al 1997, Kim et al 1998, Velankar et al 1999) we propose the following working model for a nucleic acid substrate binding by dengue NS3: 171—618 (Fig. 5). Translocation in the 3' ^ 5' direction along a single-stranded nucleic acid tail of about 6—8 (ribo) -nucleotides, trapped in the tunnel, would result from interdomain movements triggered by the hydrolysis of a nucleotide at the NTP binding site. The duplex portion of the substrate would contact the concave surface between domains II and III (Fig. 5).

Structure-based site directed mutagenesis

Based on the 3D structure, we carried out site-directed mutagenesis to probe the function of a number of residues for ATPase activity and duplex unwinding. Fourteen mutations were introduced within the helicase domain of the NS3 protein and each mutated protein was tested for its ATPase, RNA helicase and RNA binding activities. A mapping of residues identified in this study that are critical

FIG. 4. Close-up view of the ATP binding pocket of dengue NS3: 171—618 (sticks) with a bound Mn2+ and a sulfate ion from the crystallization solution. Residues are labelled. The divalent Mn2+ (represented as a dark grey sphere) is coordinated by residues Glu285 and four water molecules.

for enzymatic activity is shown in Fig. 6. We identified four residues which selectively disrupt either the ATPase (Arg460, Arg463) or helicase (Ile365, Arg376) activity of the full-length protein, when individually mutated to alanine (Fig. 6). The sole substitution into alanine of Lys396 which is located at the surface of domain II resulted in a NS3 protein having lost both enzymatic activities. We propose that residue Ile365 which is found at the tip of domain II and lines the putative single-strand RNA binding tunnel at a position closed to the fork (Fig. 5), is crucial for translocation of the enzyme along the nucleic acid substrate.

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