title: The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.
abstract: Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, 01605.
authors.forename: Cihan
authors.initials: C
authors.lastname: Aydin
authors.forename: Sourav
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Hanson
authors.forename: David N
authors.initials: DN
authors.lastname: Frick
authors.forename: Celia A
authors.initials: CA
authors.lastname: Schiffer
mesh_terms: D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D016174:Hepacivirus / Q000201:enzymology* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D064251:Multifunctional Enzymes / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D004265:DNA Helicases; D047908:Intracellular Signaling Peptides and Proteins; D064251:Multifunctional Enzymes; D010447:Peptide Hydrolases; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus
keywords: HCV NS3/4A; bifunctional enzyme; catalytic activity; dynamic coupling; interdomain communication; protease-helicase interaction