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.

Keywords: HCV NS3/4A; bifunctional enzyme; catalytic activity; dynamic coupling; interdomain communication; protease-helicase interaction.

Figure 1

(A) Cartoon representation of HCV scNS3/4A (PDB ID: 1CU1). In this structure, helicase domain covers the otherwise solvent exposed active site of the protease and the C-terminus of the protein occupies the P-side of protease active site. (B) Close-up view of the domain–domain interface. (Left) The substrate interface is formed between the C-terminus (yellow), and residues from the helicase (orange) and the protease. (Right) The direct interface is formed by directly contacting residues between the protease (blue) and the helicase (red). These two interfaces lie on either side of the catalytic triad (magenta).

Figure 2

(A) (Top) Visualized cross correlations for wildtype protein and in silico deletion constructs. Correlations between 0.35 and 0.45 were colored yellow, larger than 0.45 red, between −0.35 and −0.45 cyan, lower than −0.45 blue and remaining gray. (Bottom) Distributions of interdomain correlations for each construct. (B) Pairwise distance difference distribution functions for selected residues across interfaces. Each residue pair contains one residue from the protease and one from the helicase. Distance differences are obtained by subtracting crystallographic distances from the simulation distances. Wildtype protein is represented with gray, ΔLin yellow, ΔC-term green, and ΔDouble orange.

Figure 3

(A) (Left) Upon cleavage of the RET-S1 substrate, the FRET pair is liberated and the increase in fluorescence intensity is recorded kinetically (black – no protein, red to green – increasing enzyme concentration). Progress curves were fit to first order rate equations to obtain specificity constants (k_s). (Right) Specificity constants at various enzyme concentrations were subjected to concatenate linear fit to yield catalytic efficiencies. (B) Substrate cleavage catalytic efficiencies of each protease variant, normalized to scNS3/4A. Error bars represent propagated standard errors (n = 6).

Figure 4

(A) DNA unwinding assay. dsDNA substrate for DNA unwinding assays consists of a top strand labeled with Cy5-IBRQ FRET pair at both ends and a longer bottom strand. Upon strand separation, the top strand self-anneals and Cy5 fluorescence is quenched. (B) (Left) Loss of fluorescence is recorded kinetically (black – no protein, red to purple – increasing protein concentration) and rates were obtained from nonlinear regression. (Right) Rates were plotted against enzyme concentration and linear parts of these curves were fitted concatenately to yield specific rates (V/E) from linear regression. (C) (Top) The difference between multiple and single turnover DNA unwinding assays. For each variant, fold change in activities for the single turnover activities with respect to multiple turnover were calculated. Error bars represent propagated standard errors (n = 3). (Bottom) Specific activities of each helicase variant for multiple turnover (bars) and single turnover (bars) DNA unwinding assays, normalized to scNS3/4A multiple turnover activity. Error bars represent propagated standard errors (n = 3). (D) RNA unwinding assay. dsRNA-DNA hybrid substrate for RNA unwinding assays consists of an RNA Cy5-labeled top strand, a DNA IBRQ-labeled top strand and a long RNA bottom strand. Upon unwinding, both top strands are liberated and self-anneal, resulting in increased Cy5 fluorescence intensity. (E) Gain of fluorescence was recorded kinetically (black – no protein, red to purple – increasing protein concentration) and data analysis were performed similar to DNA unwinding assay. (F) Specific activities of each helicase variant for RNA unwinding assays, normalized to scNS3/4A. Error bars represent propagated standard errors (n = 4).

Figure 5

(A) Dissociation constants (K_D) for DNA binding, normalized against scNS3/4A. (B) (Top) RNA-dependent catalytic rate constants (k_cat) and (Bottom) extent of ATPase stimulation by RNA (K_RNA) for all helicase variants, normalized to scNS3/4A. Error bars represent propagated standard errors (n = 3).

Figure 6

Possible models unifying our experimental results and previous observations on relations between HCV NS3/4A conformational states and protease and helicase function. (A) Binding of the P-side of a substrate induces conformation change from the closed conformation to a semiextended conformation. Conformational switch from the proposed semi-extended conformation to the extended conformation depends on the stability of the direct interface and the protease is fully active in the extended conformation. (B) Binding to the single-stranded region of a nucleic acid (either DNA or RNA) occurs in the closed conformation. For dsDNA, activation of the protein was modulated by interface mutations significantly, possibly due to the alteration of the dynamic interaction between the domains through the protease-helicase interface. For dsRNA, activity is independent of the interface, and protease-RNA association acts a factor modulating the conformational transition. The protease domain potentially associates with the dsRNA as a clamp. When the protein oligomerizes on dsRNA, two possible arrangements for RNA-protease interaction is possible – either all the protease domains or only the domain on the leading monomer associate with dsRNA.