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Nucleic Acids Research 2004 32(18):5519-5528; doi:10.1093/nar/gkh891
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Published online 12 October 2004

Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved

Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations

David N. Frick*, Ryan S. Rypma, Angela M. I. Lam and Christopher M. Frenz

Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA

* To whom correspondence should be addressed. Tel: +1 914 594 4190; Fax: +1 914 594 4058; Email: David_Frick{at}NYMC.edu

Received August 11, 2004; Revised September 16, 2004; Accepted September 27, 2004

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pKas. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pKas in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pKa values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pKas including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pKas shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.


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