The helicase catalytic domain of NS3 displays strand displacement activity using both dsDNA and dsRNA as substrates (Xu et al 2005). We compared the unwinding activity of the truncated NS3 protein with the full length NS3 protease/heli-case (NS3FL). Interestingly, NS3FL shows a significantly higher unwinding activity compared to the helicase domain. In the absence of a structure for the full length NS3 protein, how the N-terminal protease domain of dengue NS3 influences the helicase activity is currently unknown. We determined the kinetic parameters for ATP hydrolysis for both NS3: 171-618 and NS3FL. Both enzymes have similar turnover numbers (kcat = 6.9 s-1 and 5.9 s-1, for NS3: 171-618 and NS3FL respectively). NS3: 171-618 has a higher affinity for ATP (Km 33 ± 3 |iM) than NS3FL (Km 297 ± 34 |iM). Thus, the NS3 fragment we crystallized is enzymatically active both in NTP hydrolysis and duplex unwinding. Enzymatic activities are likely to be further regulated by additional protein-protein interactions in the context of the viral replication complex.
The structure of dengue NS3 helicase (Fig. 1) comprises three domains of equal dimensions. Domain I (residues 181—326) and domain II (residues 327—481) show little sequence identity with each other, but are structurally similar. They feature a large central six-stranded parallel P-sheet, flanked by four a-helices. Domain III (residues 482—618) is composed of four approximately parallel a-helices (a/', a3", a4", a7"), surrounded by three shorter helices (02", a5", a6"), and augmented by two
anti-parallel P-strands largely exposed to the solvent. A long p-hairpin (p4A', p4B') extends from domain II into domain III. A tunnel runs across the centre of the most basic face of the protein. A side-by-side comparison between the Den NS3: 171—618 and the HCV NS3 helicase structures highlights the conservation of the tandem core structure while the third terminal domain bears no structural similarity (Fig. 2), an observation consistent with the pattern of amino-acid sequence identities between the individual domains of den NS3 and their counterparts in HCV (Xu et al 2005).
A superposition with the yellow fever virus helicase (Wu et al 2005) reveals possible hinge motion between domain III and the RecA tandem repeat, as well as movements in helices al' and a7' (Fig. 3). Recently, residues 303—618 of dengue NS3 were shown to bind to the RNA dependent RNA polymerase NS5 (Brooks et al 2002). This interaction might involve the C-terminal domain III of dengue NS3. Indeed, a major challenge for future investigations consists in the precise mapping of the intermolecular interactions that take place within a functional viral replication complex.
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