Mechanistic insights into Zika virus NS3 helicase inhibition by Epigallocatechin-3-gallate: Supplementary Files

Deepak Kumar; Nitin Sharma; Murali Aarthy; Sanjeev Singh; Rajanish Giri

Molecular dynamics simulation studies help in understanding the protein structurefunction such as folding, conformational flexibility, and stability. Hence, we have performed the MD simulations on the apo ZIKV helicase and compared the protein stability when EGCG bound at NTPase site and RNA binding site for a period of 100ns ( Figure 3 and Figure 4 respectively). In figure 3A , the analysis of C-α root mean square deviations (RMSD) portrays that the apo ZIKV helicase represented in black did not seem to be stable enough (RMSD =1.75 -2.75 Å) throughout the simulation when compared to the EGCG-NTPase complex represented in red (RMSD = 1.5 Å to 2.25 Å). Initially the EGCG complex was showing similar fluctuations like apo helicase but after 65 ns the complex was observed to achieve its stability till the completion of simulation course period. In order to understand the conformational fluctuations upon EGCG binding at NTPase pocket, C-α root mean square fluctuation (RMSF) at single residue level were compared between apo helicase and complex ( Figure 3B ). In Figure 3B , it was observed that the region around 248-255 was showng higher fluctuations of 4.75 Å in EGCG complex and lower fluctuations of 2.75 Å were monitored in region 320-326 in EGCG complex as compared to apo helicase. Notably, studies have shown that the region 248-255 belongs to RNA binding loop (244-255) which is highly dynamic and stabilized after RNA binding (21, 32) . Interestingly, it was noticed that P-loop region (193-203) was not showing fluctuations in EGCG complex and apo helicase as well. Since dynamic P-loop is critical for ATP hydrolysis, it may be concluded that EGCG stabilizes the P-loop dynamics by forming significant interactions with key residues as observed in simulation interaction diagram ( Figure S1C ). In Figure 3C , compactness of the protein structure was measured by comparing the radius of gyration (R g ) in apo helicase and EGCG complex. It was observed that the compactness of protein was maintained stably throughout 100ns simulation period in EGCG complex as compared to apo helicase. This also shows that the dynamic NTPase site is also controlling the overall shape of protein and EGCG is probably stabilising the NTPase site that further maintaining the overall compactness. Further, the analysis of secondary structure elements ( Figure S1A ) revealed that the region 180-185, 300-310, 330-350 and 450-470 shows unstable conformation throughout the simulation time. More deeply, in figure S1B the timeline of percentage index of total contacts was represented and analysis revealed that ARG 462, THR 201, GLU 286 and ARG 459 had retained the contacts throughout simulation period. In figure 3D , the interaction histogram in combination with 2D simulation interaction diagram ( Figure S1D ) was showing fraction of different interactions (H-bond, hydrophobic, ionic and water bridges) between EGCG and NTPase site residues. It was observed that residues GLU 286 and THR 201 have ionic interaction contribution of 100 %, while ARG 462, ARG 459 and GLY 199 shows the hydrogen bond interaction of more than 70% of simulation time. Figure 1C was showing maintenance of Hbonding throughout the simulation.