The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities
Department of Biochemistry and Molecular Pharmacology
Biochemistry | Molecular Biology
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.
HCV NS3/4A, bifunctional enzyme, catalytic activity, dynamic coupling, interdomain communication, protease-helicase interaction
Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M.; Frick, David N.; and Schiffer, Celia A., "The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities" (2013). University of Massachusetts Medical School Faculty Publications. 260.