The protease domain increases the translocation stepping efficiency of the hepatitis c virus NS3-4A helicase

Vaishnavi Rajagopal, Madhura Gurjar, Mikhail K. Levin, Smita S. Patel

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

Hepatitis C virus (HCV) NS3 protein has two enzymatic activities of helicase and protease that are essential for viral replication. The helicase separates the strands of DNA and RNA duplexes using the energy from ATP hydrolysis. To understand how ATP hydrolysis is coupled to helicase movement, we measured the single turnover helicase translocation-dissociation kinetics and the pre-steady-state Pi release kinetics on singles-tranded RNA and DNA substrates of different lengths. The parameters of stepping were determined from global fitting of the two types of kinetic measurements into a computational model that describes translocation as a sequence of coupled hydrolysis-stepping reactions. Our results show that the HCV helicase moves with a faster rate on single stranded RNA than on DNA. The HCV helicase steps on the RNA or DNA one nucleotide at a time, and due to imperfect coupling, not every ATP hydrolysis event produces a successful step. Comparison of the helicase domain (NS3h) with the protease-helicase (NS3-4A) shows that the most significant contribution of the protease domain is to improve the translocation stepping efficiency of the helicase. Whereas for NS3h, only 20% of the hydrolysis events result in translocation, the coupling for NS3-4A is near-perfect 93%. The presence of the protease domain also significantly reduces the stepping rate, but it doubles the processivity. These effects of the protease domain on the helicase can be explained by an improved allosteric cross-talk between the ATP- and nucleic acid-binding sites achieved by the overall stabilization of the helicase domain structure.

Original languageEnglish (US)
Pages (from-to)17821-17832
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number23
DOIs
StatePublished - Jun 4 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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