2.2.3. Molecular Docking

To confirm the edG-HS interaction, we performed the molecular docking experiment and estimated the binding energy, intramolecular distance and interacting residues of protein with the heparan sulfate. This analysis evaluated the binding interaction of heparan sulfate with the edG and helped us understand its role as an attractive target. Figure 8A shows the protein (surface view) heparan sulfate (ball and stick) interaction with various amino acid residues. The docking study suggested that heparan sulfate occupies the active binding site of the edG with a strong binding affinity of −6.8 kcal/mol. The protein–ligand complex forms the hydrogen bond interactions with seven key residues: Asn112, Lys117, Thr133, Thr134, Ser191, Arg196, and Glu226 (Figure 8A). Figure 8B shows the bond distance between the ligands (HS) with the interacting amino acid residues of the protein. The HS interact via a single bond with six amino acids viz. Asn112 of distance 2.04 Å, Lys117 of 2.74 Å, Thr133 of 2.92 Å, Thr134 of 2.03 Å, Ser191 of 2.64 Å, Arg196 of 2.27 Å, and via two bonds with Glu226 with an equal distance of 2.13 Å. The distance of the hydrogen bond lies in the range of 2.03–2.92 Å, which indicates the good binding affinity between the protein–

ligand complex. In addition, the protein–ligand complex also forms four carbon–hydrogen bonds with key residues of Leu115, Cys116, Pro132, and Arg196 depicted in the 2D model (Figure 8C). The higher number of hydrogen bonds and lower binding energy suggests the strong binding affinity of the protein–ligand complex.

**Figure 8.** Interaction of edG with heparan-sulfate. (**A**) Surface representation of docked edG-HS complex in the active site pocket and important residues involved in the polar interactions (stick model). (**B**) Representation of edG-HS complex with the distance of hydrogen bonds. (**C**) Detailed 2-dimensional representation showing interactions and types of bonds formed between heparan sulfate and edG.

Several viruses use heparan sulfate proteoglycans (HSPGs) on the cell surface as attachment factors such as vaccinia virus [58], herpes simplex virus [59], hepatitis C virus [60], Sindbis virus [61], human immunodeficiency virus-1 (HIV-1) [62] and HCoV-NL63 [63]. Recently a study reported that the spike protein of SARS COV-2 interacts with heparan sulphate and ACE2 through RBD and promotes the spike–ACE2 interaction [22]. A study by Kalia et al. reported that HS plays a critical role in facilitating HEV infection on target cells because the elimination of heparan sulfate by heparinase hindered pORF2 attachment and blocked infection of HEV to Huh-7 cells [64]. Another study has reported the interaction of ectodomain G protein with quercetin and morin (flavonoids) utilizing fluorescence quenching and suggested it is an antiviral agent against RSV [65]. In our study, we found that the number of hydrogen bonds formed by edG with heparan sulfate is significantly high and hence have an excellent binding affinity. The binding results obtained from fluorescence quenching complement our molecular docking observation that attributed that conventional hydrogen bond and carbon-hydrogen bond was mainly widespread in the protein–ligand complex. The binding parameter of edG–heparan sulfate complex

obtained from fluorescence quenching, molecular docking, and previously reported results from ITC and MST [30] are given in Table 3.

**Table 3.** Binding energy parameters of heparan sulfate with edG obtained from fluorescence and docking studies.


#K calculated from fluorescence quenching, \*∆G calculated from molecular docking, @K calculated from ITC, \*K*<sup>d</sup>* calculated from MST.
