Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations. Nucleic Acids Res 2004;32(18):5519-28
Date
10/14/2004Pubmed ID
15479787Pubmed Central ID
PMC524300DOI
10.1093/nar/gkh891Scopus ID
2-s2.0-6044262451 (requires institutional sign-in at Scopus site) 32 CitationsAbstract
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 pK(a)s. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK(a)s 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 pK(a) 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 pK(a)s 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 pK(a)s shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.
Author List
Frick DN, Rypma RS, Lam AM, Frenz CMAuthor
David N. Frick PhD Associate Professor in the Chimistry & Biochemistry department at University of Wisconsin - MilwaukeeMESH terms used to index this publication - Major topics in bold
Allosteric SiteBinding Sites
DEAD-box RNA Helicases
Hydrogen-Ion Concentration
Mutagenesis, Site-Directed
Nucleoside-Triphosphatase
Protein Conformation
RNA Helicases
Serine Endopeptidases
Static Electricity
Viral Nonstructural Proteins
