Faculty Collaboration Database

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities. Protein Sci 2013 Dec;22(12):1786-98

Date

10/15/2013

Pubmed ID

24123290

Pubmed Central ID

PMC3843632

DOI

10.1002/pro.2378

Scopus ID

2-s2.0-84892910710 (requires institutional sign-in at Scopus site)   18 Citations

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.

Author List

Aydin C, Mukherjee S, Hanson AM, Frick DN, Schiffer CA

Author

David N. Frick PhD Associate Professor in the Chimistry & Biochemistry department at University of Wisconsin - Milwaukee

MESH terms used to index this publication - Major topics in bold

Amino Acid Substitution
Carrier Proteins
DEAD-box RNA Helicases
DNA Helicases
Hepacivirus
Intracellular Signaling Peptides and Proteins
Kinetics
Molecular Dynamics Simulation
Multifunctional Enzymes
Nucleoside-Triphosphatase
Peptide Hydrolases
Protein Binding
Protein Conformation
Protein Structure, Tertiary
RNA Helicases
Serine Endopeptidases
Viral Nonstructural Proteins