2.2.4. MD Simulation Studies

We performed extensive MD simulation studies to know the interaction mechanism of edG-HS complex and edG alone for 50 ns. We assumed that HS has a close binding conformation with edG. The system stability was determined by calculating the radius of gyration (Rg) and root-mean-square deviations (RMSD) values, which showed that after ~20 ns, the system achieved the equilibrium conformation (Figure 9A,B). A clear difference in the R<sup>g</sup> and RMSD values of edG and edG-HS complex suggested that complex form showed higher dynamic value than the unbound form, which can be attributed to the perturbation effect of HS on the structure of edG. The active binding site (CX3C motif and heparin-binding region) of edG lies in the amino acid region 110–130. The root mean square fluctuation (RMSF) plot of the edG and edG-HS complex showed a considerable variation in the structure of these residues (Figure 9C). Compared to the edG-HS complex, the active site residues in the free edG showed a lower degree of mobility in the constituents' residues, indicating lower relative energy. The solvent-accessible surface area (SASA) plot showed no significant changes throughout the 50 ns simulation process, attributing that formation of a stable complex between edG and HS (Figure 9D). The SASA value for edG-HS complex and edG alone was found to be 127 nm<sup>2</sup> and 130 nm<sup>2</sup> , respectively.

**Figure 9.** Structural dynamics, compactness and folding of the edG-HS complex as a function of time. (**A**) RMSD plot of edG and edG-HS complex. (**B**) The Rg curves of edG and edG-HS complex showing differences in compactness. (**C**) RMSF plot of edG and edG-HS complex. (**D**) SASA plot of edG before and after binding with HS as a function of time.

The average intermolecular hydrogen bonds were ~3.0 between HS and protein during the MD simulation process (Figure 10A). Furthermore, the total energy presented between edG alone and edG-HS complex was calculated using GROMACS utility (Figure 10B). The total free energy of edG was found −768 kJ/mol, and the edG-HS complex was found −772 kJ/mol, with electrostatic energy accounting for a significant contribution. The structural conformation difference between the edG and edG-HS complex was also studied by analyzing the free energy landscape. No major conformation difference was observed between protein and protein–HS complex (Figure 10C,D). The edG showed an energyfavoured and relatively stable conformation compared to the protein–ligand complex, suggesting that binding of HS not perturbed the structure of the protein.

**Figure 10.** (**A**) The fluctuating curve of hydrogen bonds shows changes in the observed number. (**B**) Generated curves of free energy landscapes showing fluctuations of total energies observed between edG-HS and edG. (**C**) Free energy landscape plot of edG and (**D**) edG-HS complex.
