ATP Binding Modulates the Nucleic Acid Affinity of Hepatitis C Virus Helicase

Abstract

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the ␤-␥ methylene/amine analogs of ATP, ATP␥S, or [AlF 4 ]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF 3 ]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3 single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.Helicases are molecular motors that use the energy of ATP hydrolysis to unwind nucleic acid. HCV 1 codes for a helicase that constitutes the C-terminal domain of NS3 protein. Here we have studied the properties of the helicase domain of NS3 protein (NS3h) expressed as a separate polypeptide. NS3h is a 3Ј to 5Ј helicase that can unwind both RNA and DNA (1, 2). Nucleic acid unwinding is a stepwise process involving translocation and local separation of the duplex nucleic acid strands (3). In the wedge mechanism, a simple mechanism of unwinding, strand separation is accomplished by unidirectional translocation of the helicase and capture of the ssDNA (3, 4). In such a mechanism, the basic activity of the helicase is unidirectional translocation driven by the ATPase cycles. The mechanism by which ATPase drives directional translocation of the helicase is however not known.Each cycle of the ATPase reaction consists of a number of steps including ATP binding, hydrolysis, phosphate release, ADP release, and rebinding of ATP.The ATPase cycle allows the helicase to adopt various nucleotide ligation states that allosterically control the conformation of the nucleic acid binding site. Thus, it is believed that the ATPase cycle causes conformational changes in the ...

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

“…As was seen previously, DNA binds HCV helicase about 10-fold weaker in the presence of ADP(BeF 3 ) (16,22). This has been interpreted to mean that ATP binding allows the protein to slide along DNA (22). Using this assay, the apparent K D for the 18-mer in the absence of ADP(BeF 3 ) is 1.9 Ϯ 0.3 nM, whereas the K D with F18 is 2.5 Ϯ 0.3.…”

“…F18 and the same oligonucleotide lacking the fluorescein (18-mer) bound similarly both in the presence and absence of the nonhydrolyzable ATP analog ADP(BeF 3 ). As was seen previously, DNA binds HCV helicase about 10-fold weaker in the presence of ADP(BeF 3 ) (16,22). This has been interpreted to mean that ATP binding allows the protein to slide along DNA (22).…”

“…Table II also summarizes the results of the same set of experiments when they were conducted in the presence of the nonhydrolyzable ATP analog ADP(BeF 3 ). Importantly, both Hel-His and His-NS3-4A appear to release ssDNA upon ATP binding, indicating that the full-length enzyme could function using the same bind-slide mechanism proposed by Levin et al (22).…”

“…Previously, intrinsic protein fluorescence has been used to examine ssDNA interaction with HCV helicase (15,16,22,35,38). All of these studies used recombinant proteins lacking the protease domain and exploit the fact that ssDNA binds near a solvent exposed tryptophan to quench intrinsic protein fluorescence (7).…”

“…DNA oligonucleotide was either the same sequence as the short strand used in unwinding assay (18-mer) 5Ј-GCC TCG CTG CCG TCG CCA-3Ј or the same oligonucleotide with a fluorescein moiety attached to its 3Ј-end (F18). Experiments were performed in the presence and absence of the nonhydrolyzable ATP analog ADP(BeF 3 ), which was formed by including 0.1 mM ADP, 5 mM NaF, and 0.5 mM BeF 2 in the reaction buffer (22). After correcting data for dilution and inner filter effects, fractional fluorescence (FF) was calculated by dividing the fluorescence values by the initial fluorescence of the protein in the absence of DNA.…”