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. 2018 Oct 30;57(43):6247-6255.
doi: 10.1021/acs.biochem.8b00796. Epub 2018 Oct 16.

Role of the Conserved DECH-Box Cysteine in Coupling Hepatitis C Virus Helicase-Catalyzed ATP Hydrolysis to RNA Unwinding

Mark M Yerukhimovich  1 , Christopher C Marohnic  2 , David N Frick  1
Affiliations

Role of the Conserved DECH-Box Cysteine in Coupling Hepatitis C Virus Helicase-Catalyzed ATP Hydrolysis to RNA Unwinding

Mark M Yerukhimovich et al. Biochemistry. .

Abstract

DECH-box proteins are a subset of DExH/D-box superfamily 2 helicases possessing a conserved Asp-Glu-Cys-His motif in their ATP binding site. The conserved His helps position the Asp and Glu residues, which coordinate the divalent metal cation that connects the protein to ATP and activate the water molecule needed for ATP hydrolysis, but the role of the Cys is still unclear. This study uses site-directed mutants of the model DECH-box helicase encoded by the hepatitis C virus (HCV) to examine the role of the Cys in helicase action. Proteins lacking a Cys unwound DNA less efficiently than wild-type proteins did. For example, at low protein concentrations, a helicase harboring a Gly instead of the DECH-box Cys unwound DNA more slowly than the wild-type helicase did, but at higher protein concentrations, the two proteins unwound DNA at similar rates. All HCV proteins analyzed had similar affinities for ATP and nucleic acids and hydrolyzed ATP in the presence of RNA at similar rates. However, in the absence of RNA, all proteins lacking a DECH-box cysteine hydrolyzed ATP 10-15 times faster with higher Km values, and lower apparent affinities for metal ions, compared to those observed with wild-type proteins. These differences were observed with proteins isolated from HCV genotypes 2a and 1b, suggesting that this role is conserved. These data suggest the helicase needs Cys292 to bind ATP in a state where ATP is not hydrolyzed until RNA binds.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
DECH-box motif in RNA helicases. The DECH-box motif in a crystal structure of HCV NS3h bound to the ground state ATP analogue ADP·BeF3 [tan, Protein Data Bank (PDB) entry 3KQN] and transition state analogue ADP·AlF4− (blue, PDB entry 3KQL). Also shown are the Mg2+ ion (tan in PDB entry 3KQL) and Mn2+ ions (blue, from PDB entry 3KQL) that connect the DECH box to ADP. Below is an alignment of the amino acids surrounding the DECH box in selected DExD/H-box proteins. Residues are colored on a red–blue gradient based on conservation (blue, conserved; red, variable). RIG-I, MDA5, and LGP2 are cytoplasmic RNA receptors that are key innate immune system components. PP C1 is an RNA helicase from plum pox virus, YFV NS3 from yellow fever virus (the namesake flavivirus), and BVDV NS3 from a pestivirus called bovine viral diarrhea virus (flavivirus, pestivirus, and hepacivirus are three genera in the Flaviviridae family).
Figure 2.
Figure 2.
Effect of the C292G substitution on helicase-catalyzed DNA unwinding. (A) Assay setup in which NS3h displaces a molecular beacon annealed to a complementary strand with a 3′ single-stranded DNA tail. The beacon and its complement form hairpins upon separation, leading to a decrease in the observed fluorescence. (B) MBHAs performed with various concentrations (nanomolar) of NS3_2a(JFH1). (C) MBHAs performed with various concentrations of NS3_2a(C292G). In panels B and C, data obtained after ATP addition (points) were fit to eq 1 (solid lines) yielding a rate constant (kobs) and amplitude (A). Initial velocities were calculated by multiplying these two values. (D) Initial velocities obtained with NS3_2a(JFH1) (○) and NS3_2a(C292G) (□) fit to eq 2 (Materials and Methods) using the Vmax and K0.5 in the table (right). Uncertainties in the table represent 95% confidence intervals for the nonlinear regression analyses performed with GraphPad Prism.
Figure 3.
Figure 3.
Effect of the C292G substitution on the affinity of NS3h for DNA and RNA. (A) The florescence polarization of Cy5′-dT15 (5 nM) was monitored in solutions containing the indicated concentrations of either NS3_2a(JFH1) (○) or NS3_2a(C292G) (□). Averages of three replicates are shown (points). Error bars are standard deviations. Data were fit to a quadratic binding equation (eq 3) with the Kd values listed in the table. (B) Turnover rates of ATP hydrolysis (micromoles of ATP cleaved per second per micromole of enzyme) observed in reaction mixtures containing either helicase (5 nM) and the indicated poly(U) RNA concentrations (in micromoles of nucleotides per liter). The initial ATP concentration in each reaction was 10 mM, and hydrolysis was monitored by measuring the concentration of orthophosphate liberated by the enzyme., Data were fit to eq 4 with the constants listed in the table. Uncertainties in the tables represent 95% confidence intervals for the nonlinear regression analyses performed with GraphPad Prism.
Figure 4.
Figure 4.
Effect of the C292G substitution on the kinetics of NS3h-catalyzed ATP hydrolysis. Helicase-catalyzed ATP hydrolysis was monitored, as described in the legend of Figure 3B, in reaction mixtures containing various amounts of MgATP2− and additional 1 mM MgCl2. (A) Rates of ATP hydrolysis observed in reaction mixtures containing either helicase (100 nM) in the absence of RNA. (B) Rates of ATP hydrolysis observed in reaction mixtures containing either helicase (5 nM) and 300 μM poly(U) RNA. Data were fit to (A) eq 5 or (B) eq 6 with the indicated values for kslow, Km, Kfast, and Km*. Uncertainties in the tables represent 95% confidence intervals for the nonlinear regression analyses performed with GraphPad Prism.
Figure 5.
Figure 5.
Role of Cys292 in the interaction of HCV helicase with divalent metal cations. Rates of NS3_2a(JFH1)- and NS3_2a(C292G)-catalyzed ATP hydrolysis were monitored in reaction mixtures containing 10 mM ATP and various concentrations of (A and C) MgCl2 or (B and D) MnCl2. Reaction mixtures for panels A and B contained 100 nM enzyme, and reaction mixtures for panels C and D contained either enzyme (5 nM). Rates are expressed as micromolar ATP cleaved per micromolar enzyme per second, and data were fit to (A) eq 7 or (C and D) eq 8 with the indicated values. Uncertainties represent 95% confidence intervals for the fit. Data in panel B did not fit eq 7 because no stimulation by MnCl2 was observed in the absence of RNA.
Figure 6.
Figure 6.
Effect of an NS3 C292S substitution in the HCV genotype 1b genetic background. (A) All cysteine residues (magenta) highlighted in a structure of the genotype 1b NS3h protein bound to DNA (blue) and the nonhydrolyzable ATP analogue ADP·AlF4 (PDB entry 3KQL). Residues targeted for site-directed mutagenesis are labeled. (B) Comparison of rates of helicase-catalyzed ATP hydrolysis at various ATP concentrations as described in Figure 4. (C) Comparison of kslow values (left axis) and Km values (right axis) obtained with each protein listed on the x-axis in assays monitoring ATP hydrolysis. In B and C, red points designate proteins harboring a C292S substitution, and in C, and error bars represent 95% confidence intervals for the fit.
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