Differences in the Membrane-Binding Properties of Flaviviral Nonstructural 1 (NS1) Protein: Comparative Simulations of Zika and Dengue Virus NS1 Proteins in Explicit Bilayers

Abstract

NS1 in flaviviruses is the only nonstructural protein that is secretory and interacts with different cellular components of the host cell membrane. NS1 is localized in the ER as a dimer to facilitate viral replication. Crystal structures of NS1 homologues from zika (ZIKV) and dengue (DENV) viruses have revealed the organization of different domains in NS1 dimers. The β-roll and the connector and intertwined loop regions of wing domains of NS1 have been shown to interact with the membranes. In this study, we have performed multiple molecular dynamics (MD) simulations of ZIKV and DENV NS1 systems in apo and in POPE bilayers with different cholesterol concentrations (0, 20 and 40%). The NS1 protein was placed just above the membrane surface, and for each NS1-membrane system two to three independent simulations with 600 ns production run were performed. At the end of the production runs, ZIKV NS1 inserts deeper inside the membrane compared to the DENV counterpart. Unlike ZIKV NS1, the orientation of DENV NS1 is asymmetric in which one of the chains in the dimer interacts with the membrane while the other is more exposed to the solvent. The β-roll region in ZIKV NS1 penetrates beyond the headgroup region and interacts with the lipid acyl chains while the C-terminal region barely interacts with the headgroup. Specific residues in the intertwined region deeply penetrate inside the membrane. The role of charged and aromatic residues of ZIKV NS1 in strongly interacting with the membrane components is revealed. The presence of cholesterol affects the extent of insertion in the membrane and interaction of individual residues. Overall, membrane-binding properties of ZIKV NS1 significantly differ from its counterpart in DENV. The differences found in the binding and insertion of NS1 can be used to design drugs and novel antibodies that can be flavivirus specific.

Figure 1

(a) Superposed NS1 dimer structures from Zika and Dengue viruses. The β-roll, wing, intertwined loop, and C-terminal domains of ZIKV NS1 are displayed in red, cyan, dark brown, and dark green, respectively. The same domains in DENV NS1 are shown in orange, blue, light brown, and light green, respectively. (b) Initial simulation setup of NS1-membrane complex. The two chains of NS1 are shown as cartoon representations in different colors. The membrane is shown in stick representation (gray color) and water molecules as surface representation (light gray).

Figure 2

Time evolution of RMSD (average of replicates) of (a) ZIKV NS1 and (b) DENV NS1. RMSD calculated for the backbone atoms of NS1 dimer (both chains A and B) with respect to the starting structure. The different NS1 complexes are differentiated as ZIKV_APO (cyan), ZIKV_POPE (blue), ZIKV_CHOL20 (magenta), ZIKV_CHOL40 (black), DENV_APO (yellow), DENV_POPE (dark green), DENV_CHOL20 (red) and DENV_CHOL40 (violet). The RMSDs of POPE and 40% cholesterol complexes are the results of average of three replicates while the RMSD of 20% cholesterol complexes are due to the average of two replicates.

Figure 3

Histograms showing the probability distributions of (a,c) distance d and (b,d) and angle θ between membrane and NS1, and these parameters were used to characterize the orientation of NS1 with respect to the membrane for both (a, b) ZIKV and (c, d) DENV NS1 systems. The histograms are plotted by calculating the averages of these parameters from the last 300 ns of the individual replicates for each system. The NS1 complexes are differentiated as ZIKV_POPE (blue), ZIKV_CHOL20 (magenta), ZIKV_CHOL40 (black), DENV_POPE (dark green), DENV_CHOL20 (red) and DENV_CHOL40 (violet). The distance d of POPE and 40% cholesterol complexes are of three replicates while the distance d of 20% cholesterol complexes is of two replicates.

Figure 4

Principal component analysis (calculated by combining the replicates) of (a–d) ZIKV NS1, (e–h) DENV NS1. The different simulation systems are colored as follows: ZIKV_APO (cyan), ZIKV_POPE (blue), ZIKV_CHOL20 (magenta), ZIKV_CHOL40 (black), DENV_APO (yellow), DENV_POPE (dark green), DENV_CHOL20 (red), and DENV_CHOL40 (violet). The PCA plot of POPE and 40% cholesterol complexes is an average of three replicates, while the PCA plot of 20% cholesterol complexes is an average of two replicates.

Figure 5

Average density profiles calculated for the individual domains of NS1 (chain A) along the Z-axis of the three different bilayers. The structure of the monomer is shown, and the respective domain used to calculate the density profiles is highlighted in orange. The different NS1 complexes are differentiated as ZIKV_POPE (blue), ZIKV_CHOL20 (magenta), ZIKV_CHOL40 (black), DENV_POPE (dark green), DENV_CHOL20 (red), and DENV_CHOL40 (violet). The POPE headgroup peak density position is indicated by the thick black line. The cholesterol headgroup peak density position is indicated by the cyan line. The zero on the X-axis corresponds to the bilayer center (as NS1 interacts with top layer and hence only top layer is shown in the figure). The profiles for individual domains are shown in rows labeled β-roll (a,e), wing (b,f), intertwined loop (c,g), and C-terminal domain (d,h) for chain A of ZIKV, DENV NS1 proteins. The density plots of POPE and 40% cholesterol complexes are an average of three replicates, while the density plots of 20% cholesterol are an average of two replicates.

Figure 6

MD simulated NS1 structures, extracted over the last 100 ns at an interval of 20 ns for all the replicates, superposed on the initial structure (gray). The intertwined loop regions are highlighted and other regions are shown as transparent. The different simulation systems are colored as follows: (a) ZIKV_APO (cyan), (b) ZIKV_POPE (blue), (c) ZIKV_CHOL20 (magenta), (d) ZIKV_CHOL40 (black), (e) DENV_APO (yellow), (f) DENV_POPE (dark green), (g) DENV_CHOL20 (red), and (h) DENV_CHOL40 (violet). Structures are shown in ribbon representation.

Figure 7

Distribution of cholesterol molecules within 4 Å of any residue (average values calculated for replicates) in ZIKV (a) and DENV (b) NS1 plotted as a probability. The different simulation systems are depicted as follows: ZIKV_CHOL20 (magenta), ZIKV_CHOL40 (black), DENV_CHOL20 (red), and DENV_CHOL40 (violet). The number of cholesterol molecules of 40% cholesterol complexes is an average of three replicates while the numbers of cholesterol molecules of 20% cholesterol complexes are an average of two replicates.