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

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

In our docking studies, we have observed that EGCG can also bind to the RNA binding cavity near entry site with significant interactions (Figure 2 and Table1). Therefore, we have run the MD simulations for apo helicase and EGCG complex at RNA binding site to compare the overall protein stability and residue level interactions (Figure 4 and Figure S2 ). The RMSD graph ( Figure 4A ) revealed that the apoprotein was showing deviation upto 20ns and attained a stable trajectory after 20ns till it reached 50ns whereas after 55ns again the deviation gradually increased by RMSD of 2.5 Ã…. In case of EGCG complex at RNA binding site, the deviation of complex was started increasing upto 2.5 Ã… till 25ns and thereafter a decrease in RMSD (2.2 Ã…) was observed and maintained throughout100ns period. This graph was showing that EGCG binding at the RNA site stabilising the overall protein conformation. In figure 4B , the comparison of RMSF was revealing that the higher fluctuations of almost 3.5 Ã… were observed in region 320-326 in apo helicase which were further reduced to 2.0 Ã… in EGCG-RNA site complex. Interestingly it was noticed that region 248-255 was showing higher fluctuations of 4.75 Ã… in EGCG complex at NTPase site ( Figure 3B ) in comparison to EGCG complex at RNA site ( Figure 4B ) which was showing similar fluctuations of 2.75 Ã… like apo helicase. Overall, the RMSF plot of EGCG-RNA site complex was found to support RMSD graph. As already mentioned before, region 248-255 contains crucial RNA binding loop which is important for helicase activity (21) . From above comparisons, it may be interpreted as EGCG binding at NTPase site may lead to the conformational changes at RNA binding loop region (244-255) which otherwise were not observed when EGCG bound at RNA site. Further in figure 4C , the radius of the gyration plot was showing that EGCG complex at RNA site had maintained compactness throughout the 100ns period as compared to apo helicase. In figure 4D , the different types of interaction fractions were observed that showed mostly H-bonded interactions were prominent between EGCG and protein. In Figure S2A , the extent of formation of secondary structure elements was analysed when EGCG bound to RNA site throughout 100ns simulation period. Further, it was noticed that H-bonding was maintained throughout the simulation ( Figure S2C ) and mostly residues GLU 413, MET 414 were continuously contacted throughout while residues LYS 431, PHE289 and ASP 410 were showing irregular contacts ( Figure S2B and S2D ). This residues observed in interaction with EGCG at RNA site, have essential role already mentioned in crystal structure of helicase with RNA (21) . For example, LYS410 and ASP410 shows important interaction with RNA sugar bases. Hence our simulation study shows that EGCG may have the capability to significantly bind at RNA site also along with NTPase site.