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Review
. 2013 Jun 26;14(7):13282-306.
doi: 10.3390/ijms140713282.

NS3 Protease From Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

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Free PMC article
Review

NS3 Protease From Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

Sonia Vega et al. Int J Mol Sci. .
Free PMC article

Abstract

The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

Figures

Figure A1
Figure A1
Stacked plots for DOSY-NMR measurements to zinc-bound NS3 protease. The exponential decay curves of NS3 shown in Figure 4 of the main text were obtained from 1D-plots as those shown in this figure, by integration of the peaks between 1.0 and 0.5 ppm. The amide region did not show any peak, apart of the aromatic residues, due to the fact that DOSY experiments were carried out in D2O, after several hours of exchange in Amicon centrifugal devices. The two different stacked plots for the two different samples of the zinc-bound NS3 are shown (see main text for the diffusion coefficients obtained). For the zinc-free NS3 similar plots were obtained. Experiments were acquired at 25 °C, at pH 5.4 (uncorrected for isotope effects).
Figure 1
Figure 1
Processing of the hepatitis C virus polyprotein. Similar to other positive-strand RNA viruses, upon infection of a hepatic cell the genomic RNA of hepatitis C virus (9.6 kb, single-stranded) serves as messenger RNA for the translation of viral proteins. The linear molecule contains a single open reading frame coding for a precursor polyprotein (~3000 aminoacid residues) consisting of 10 proteins that must be cleaved in order to be functional.
Figure 2
Figure 2
(A) Full-length NS3 protein from hepatitis C virus (PDB code: 1CU1) [26]. The dashed rectangle delimitates the protease domain (dark cyan). The zinc ion is shown as a yellow sphere. The NS4A cofactor-mimicking peptide incorporates into the NS3 protease domain as an additional beta strand (cyan); (B) NS3 protease domain in the absence of NS4A cofactor (PDB code: 1BT7) [27]. The zinc ion is shown as a yellow sphere. The zinc-coordinating residues and the catalytic residues are shown as dark cyan sticks; (C) NS3 protease domain in the presence of a NS4A cofactor-mimicking peptide (blue sticks) (PDB code: 1JXP) [28]. The zinc ion is shown as a yellow sphere. The zinc-coordinating residues and the catalytic residues are shown as dark cyan sticks. The NS4A cofactor-mimicking peptide incorporates into the NS3 protease domain as an additional beta strand (blue sticks). Comparison between (B) and (C) reveals a structural rearrangement affecting the N-terminal domain of NS3 protease upon NS4A binding and propagating to the catalytic triad (for example, D81 is reoriented upward, towards a productive conformation).
Figure 3
Figure 3
Thermodynamic dissection of the NS3 protease-zinc interaction. Temperature dependence of the Gibbs energy (ΔG, closed squares), enthalpy (ΔH, open squares) and entropy (−TΔS, open circles) for zinc binding to NS3 protease determined by ITC at pH 5. The lines correspond to the global non-linear regression fits for the temperature dependency of the Gibbs energy and enthalpy of interaction, considering a constant binding heat capacity. The strong temperature dependencies of enthalpy and entropy of binding suggest a considerable structural rearrangement coupled to metal ion binding.
Figure 4
Figure 4
DOSY-NMR measurements to NS3. The exponential decay curves of NS3 in the absence (blank squares, continuous line) and in the presence of zinc (filled squares, dotted line). The units on the y-axis are normalized intensity of the up-field shifted signals. Experiments were acquired at 25 °C, at pH 5.4 (uncorrected for isotope effects). The errors in the intensities are less than 5%, as judged from the intensity of the signal-to-noise ratio in regions of the spectra where no signals are present. The intensities for both proteins were measured by integrating in 1D-NMR experiments the most up-field shifted resonances (see Appendix).
Figure 5
Figure 5
Topography TM-AFM images and Z-height profiles associated to the lines on selected features (any individually identified element in the images) of NS3 samples. (A) NS3 monomers in presence of zinc and Z-height profile on a single monomer amplified in (B) using the zoom WSXM function [60]; (C) NS3 monomers in the absence of zinc and Z-height profile on a single monomer amplified in (D); (E) Monomers and dimer found in zinc containing samples; Z-height profile associated to the dimer and (F) image of the dimer showed in detail; (G) Monomers and dimer found in samples with no zinc; Z-height profile associated to the dimer and (H) image of the dimer shown in detail. Numbers 1 and 2 indicate the corresponding monomers composing the dimers. The 2D images show scanned areas of 100 × 100 nm, meanwhile the areas of the 3D images were chosen to show the isolated feature in detail.
Figure 6
Figure 6
Height distributions of the NS3 molecules obtained from Z-height profiles on single monomers from TM-AFM images. The data are fitted to a Gaussian model. There is a peak centred in 4.2 ± 0.5 nm in the sample containing zinc (red bars). The sample with no zinc showed a peak centred in 2.0 ± 0.3 nm (black bars). The error can be attributed to the sub-nm accuracy of the technique in fluid and the different orientations of the molecules on the mica surface.
Figure 7
Figure 7
Structural stability of NS3 protease and modulation by zinc. Thermal denaturation scans of NS3 protease followed by differential scanning calorimetry (DSC) at pH 5 (continuous lines). Excess molar heat capacity is represented as a function of temperature. Protein concentration was 40 μM, and total zinc concentration was 0 and 40 μM. The NS3 protease exhibits the following thermal stability parameters: (mid-transition temperature of 30 °C, unfolding enthalpy of 18 kcal/mol, unfolding heat capacity of 1.2 kcal/K·mol). For comparison, simulated thermal denaturation scans (see Appendix) for a protein with similar molecular mass (mid-transition temperature of 60 °C, unfolding enthalpy of 70 kcal/mol, unfolding heat capacity of 2 kcal/K·mol) exhibiting a small conformational change coupled to ligand binding, in the absence and the presence of the ligand with similar affinity to that of zinc for NS3 protease (dissociation constant of 0.5 μM), are shown (dashed lines). Although the binding affinities for zinc and that hypothetical ligand are the same for their respective binding proteins, the extent and the magnitude of the stabilization are considerably different.
Figure 8
Figure 8
Schematic depiction of the conformational landscape of NS3 protease at 20 °C considering its intrinsic structural stability and its interactions with its different ligands: zinc, NS4A and substrate. States are populated according to their Gibbs energy. Ligand (zinc, NS4A, substrate) binding modulates and shifts populations depending on the ligand binding affinity (and free ligand concentration, also). In the absence of zinc, the energetic gap between the fully unfolded state and the unstructured native state is very small (~0.4 kcal/mol), and the fully structured native state is hardly populated (high Gibbs energy). Binding of zinc, NS4A and substrate reduces the Gibbs energy of the protein. Because the binding affinity of the substrate is larger than that of NS4A ([25,62] and unpublished data), the binding of substrate stabilizes (lowers the Gibbs energy and increases the population) NS3 protease to a larger extent. States with very low population due to energetic penalty, such as the zinc-free NS4A-bound protease, are not shown.

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