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

• Published in: The Journal of biological chemistry Vol. 278; no. 26; pp. 23311 - 23316
• Main Authors: Levin, Mikhail K, Gurjar, Madhura M, Patel, Smita S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 27-06-2003
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Allosteric Regulation; Binding Sites; DNA - metabolism; DNA, Single-Stranded - metabolism; Hepacivirus - enzymology; Kinetics; Models, Molecular; Molecular Motor Proteins; Nucleic Acids - metabolism; Protein Conformation; RNA - metabolism; Spectrometry, Fluorescence; Viral Nonstructural Proteins - antagonists & inhibitors; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M301283200

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 β-γ methylene/amine analogs of ATP, ATPγS, 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.