Figure 1.
Structural model of HCV NS3/4A protein, including protein domains helicase (blue) and protease (red), cofactor NS4A (green) and the allosteric inhibitor 4VA (gray).
The C-terminal β-strand of HCV NS3 helicase domain (amino acids 626–631) is shown in orange.
Table 1.
Summary of the simulation systems.
Figure 2.
Global properties for molecular dynamics simulations.
(A) The backbone atom root mean square deviation (RMSD) for apo, inhibitor bound, apo (truncated) and inhibitor bound (truncated) HCV NS3/4A protein, calculated with respect to the initial structure during the 100 ns molecular dynamics simulation. (B) Root mean square fluctuations (RMSF) of Cα for apo, inhibitor bound, apo (truncated) and inhibitor bound (truncated) HCV NS3/4A protein averaged over the simulation. (C) RMSD of the backbone atoms of residues 101–172 and 331–420 with respect to the first snapshot as a function of time.
Figure 3.
Principal components analysis to the Cα atom motions of the simulation.
(A–D) The cloud represents the four 100 ns trajectories projected onto the first two eigenvectors. (E–H) The cloud represents the four equilibrium 20 ns trajectories projected onto the first two eigenvectors. The clouds colored in black, red, blue and, green display apo, inhibitor bound, apo (truncated) and inhibitor bound (truncated) HCV NS3/4A protein, respectively.
Figure 4.
Hydrogen bond network between the C-terminal β-strand of helicase and protease active site in HCV NS3/4A protein (apo).
The protease and helicase domains are shown in cartoon and colored in gray. The six C-terminal residues of the helicase and the protease active site residues are represented by orange and red sticks, respectively. Green dashed lines represent the hydrogen bond.
Table 2.
Analysis of hydrogen bond network between HCV NS3/4A helicase and protease domains.
Figure 5.
Distance between the backbone centers of mass of the helicase residues 614–625 and protease residues 103–171, as it varies during apo, inhibitor bound, apo (truncated) and inhibitor bound (truncated) HCV NS3/4A protein simulation.
Figure 6.
Structural models and per-residue interaction spectrums of the residues of (A–B) HCV NS3/4A protein and (C–D) its truncated protein with the allosteric inhibitor.
The representative structures extracted from the simulation trajectories were used. The proteins are shown in cyan cartoon. The inhibitor is shown in gray stick model. The residues with an absolute value larger than 0.5/mol for the inhibitor-residue interactions are shown.
Figure 7.
The polar and nonpolar interaction energies between HCV NS3/4A protein (A–B) and its truncated protein residues (C–D) with the allosteric inhibitor.
Table 3.
Binding free energy and the contributions of for the inhibitor in HCV NS3/4A proteina.
Figure 8.
Protein residue interaction network and its communities of the (A) apo (truncated) and (B) inhibitor bound HCV NS3/4A protein.
Different views of the corresponding RIN are displayed. The edges are colored with respect to their interaction type: backbone (black); interaction between closest atoms (blue); hydrogen bond (red); salt bridge (cyan); π-cation interaction (green); π-π interaction (gray). Closeness centrality denoted by nodes color (high values to bright colors). Betweenness centrality denoted by nodes size (high values to large sizes).
Table 4.
Summary of the shortest path betweenness and closeness centrality of selected residues in the network of HCV NS3/4A protein.
Figure 9.
Dynamical cross-correlation maps illustrating the correlation of motion between residues in (A) apo, (B) inhibitor bound, (C) apo (truncated) and (D) inhibitor bound (truncated) HCV NS3/4A protein.
The color scale is represented on the right ranging from red to blue: highly positive correlations are in red, highly negative correlations are in blue.
Figure 10.
Porcupine plots of the eigenvectors for simulation of apo HCV NS3/4A protein (truncated).
The model is shown as a backbone trace. The arrows attached to each backbone atom indicate the direction of the eigenvector and the size of each arrow shows the magnitude of the corresponding eigenvalue. Regions of HCV NS3/4A helicase residues 614–625 and protease residues 103–171 involved in domain movement are highlighted with blue and red, respectively.
Figure 11.
The proposed allosteric mechanism for the regulation of HCV NS3/4A protein function.
The helicase domain and the six C-terminal residues of the helicase domain, and the protease domain are represented by boxes and oval. They were colored in blue, red and orange, respectively. The allosteric inhibitor is shown as green round. The arrows in black show the C terminus of the helicase domain occupies the protease active site with and without an inhibitor binding at the allosteric site, stabilizing the protein in a closed conformation. Reactions shown with red arrows are the processes we found in this study. We found that HCV NS3/4A protein possess an extended conformation with the C-terminal residues 626–631 of the helicase domain removed and without the inhibitor binding. And with the inhibitor binding at the allosteric site, it can stabilize the closed conformation of the protein in an inactive state.