The protease domain increases the translocation stepping efficiency of the hepatitis C virus NS3-4A helicase

Abstract

Hepatitis C virus (HCV) NS3 protein has two enzymatic activities of helicase and protease that are essential for viral replication. The helicase separates the strands of DNA and RNA duplexes using the energy from ATP hydrolysis. To understand how ATP hydrolysis is coupled to helicase movement, we measured the single turnover helicase translocation-dissociation kinetics and the pre-steady-state P(i) release kinetics on single-stranded RNA and DNA substrates of different lengths. The parameters of stepping were determined from global fitting of the two types of kinetic measurements into a computational model that describes translocation as a sequence of coupled hydrolysis-stepping reactions. Our results show that the HCV helicase moves with a faster rate on single stranded RNA than on DNA. The HCV helicase steps on the RNA or DNA one nucleotide at a time, and due to imperfect coupling, not every ATP hydrolysis event produces a successful step. Comparison of the helicase domain (NS3h) with the protease-helicase (NS3-4A) shows that the most significant contribution of the protease domain is to improve the translocation stepping efficiency of the helicase. Whereas for NS3h, only 20% of the hydrolysis events result in translocation, the coupling for NS3-4A is near-perfect 93%. The presence of the protease domain also significantly reduces the stepping rate, but it doubles the processivity. These effects of the protease domain on the helicase can be explained by an improved allosteric cross-talk between the ATP- and nucleic acid-binding sites achieved by the overall stabilization of the helicase domain structure.

FIGURE 1.

The model of helicase translocation along single-stranded nucleic acid. The helicase molecules are shown as small filled triangles bound at discrete positions on the ssNA substrate; the helicase-binding site, however, spans many nucleotides. The helicase bound to the ssNA hydrolyzes ATP at a rate constant k_S. Each ATP hydrolysis event is accompanied by a translocation step. The size of each translocation step is a random integer with a mean μ. Helicase steps beyond the substrate's boundaries result in protein dissociation from the ssNA. The free helicase molecules hydrolyze ATP at a rate constant k_atp and, in the absence of a trap, re-binds the ssNA with a bimolecular rate constant k_on.

FIGURE 2.

Steady-state ATPase rate constant as a function of ssNA lengths. The ATPase rate constants of NS3h (A and B) and NS3-4A (C and D) were measured by a radiometric assay as described under “Experimental Procedures” in the presence of a 10-fold molar excess of ssDNA (A and C) or ssRNA (B and D). The ATPase rate constants are plotted as a function of substrate length. The ATPase rate constants at zero ssNA length are the intrinsic activity in the absence of ssNA. The ATPase rate constants measured in the presence of poly(dT) and poly(rU) are plotted against the average length of the polymers reported by the manufacturer.

FIGURE 3.

Single-turnover dissociation kinetics of NS3h from ssDNA and ssRNA of different lengths. A, time-dependent increase in fluorescence of NS3h due to dissociation from dT14 ssDNA (gray dots) fit into an exponential equation (black line) with a rate of 0.43 ± 0.04 s⁻¹. B, the observed NS3h exponential dissociation rates plotted as a function of ssDNA length. C, the inverse of the dissociation rate, the retention time, is plotted as a function of ssDNA length. D, time-dependent increase in fluorescence of NS3h due to dissociation from rU15 ssRNA (gray dots) fit into exponential equation (black line) with a rate of 2.11 ± 0.06 s⁻¹. E, the observed NS3h exponential dissociation rates plotted as a function of ssRNA length. F, the retention time versus ssRNA length.

FIGURE 4.

Single-turnover dissociation kinetics of NS3-4A from ssDNA and ssRNA of different lengths. A, plot of the protein dissociation rates as a function of increasing ssDNA length. B, the retention time versus ssDNA length. C, plot of the protein dissociation rates as a function of ssRNA length. D, the retention time as a function of ssRNA length.

FIGURE 5.

Effect of nucleic acid length on pre-steady-state ATPase kinetics. P_i release kinetics was measured in the presence of ssNA substrates of different length. A–C show data obtained with NS3h helicase and ssDNA substrates. D–F show data obtained with NS3h and ssRNA substrates. G–I show data for NS3-4A helicase and ssRNAs. A, D, and G show concentrations of P_i normalized by helicase concentration plotted as a function of time. The traces were fit into Equation 3, and the exponential burst rate constants k (triangles) and their amplitudes a (circles) are shown in B, E, and H as a function of substrate length. C, F, and I show reciprocals of the exponential rates, 1/k, the retention time, as a function of substrate length.