Dataset Information

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.

SUBMITTER: Aydin C

PROVIDER: S-EPMC3843632 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

Aydin Cihan C Mukherjee Sourav S Hanson Alicia M AM Frick David N DN Schiffer Celia A CA

Protein science : a publication of the Protein Society 20131019 12

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 communi ...[more]

PMID: 24123290

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Project description:The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ?2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

Dataset Information

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

Publications

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

Similar Datasets

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