## 5 Conclusions  
In the present work, we performed all-atom classical MD simulations in explicit solvent of human GSTA1 enzyme in the three different following forms of the protein: (i) the APO form, when no ligand is bound to hGSTA1, (ii) the GSH form, when the glutathione ligand is bound to the G site of hGSTA1, and (iii) the GS-R form, when the glutathione-S-conjugate is bound to the G and H sites of hGSTA1. From MD runs, we performed a free-energy landscape analysis of internal coordinates along the amino-acid sequence of the protein. The analysis was conducted using coarse-grained angles \((\theta,\gamma)\) of the main-chain (CGAs) and side-chain angles \(\chi\) (SCAs), as local probe to track conformational changes. The comparison between each form of hGSTA1 reveals 12 CGAs and 19 SCAs influenced by the ligand binding into the G and H sites. It corresponds to a total of 33 residues of hGSTA1 which are involved in the protein-ligand binding process. Among the 33 residues identified, 11 of them belong to the two binding sites of hGSTA-1 identified experimentally from XRD structures, which confirms that the method developed and applied here is very robust. Finally, the dynamics of coarse-grained and side-chain angles were studied using principal component analysis. It shows an asymmetrical behavior between the two monomers of hGSTA1, particularly from CGAs. Moreover, the analysis of the first collective motions reveals a strong dynamical coupling between residues Arg15-Gln54-Val55 of the G site and Gly14 of the H site. These residues are coupled in the same binding site (intrasite) but also between the two binding sites of each monomer of hGSTA1 (intersites), which is an important result of the present work. Finally, residue Glu17 shows important coupling with residues Arg155 and Leu163, depending on the binding form of hGSTA1 (APO and GS-R, respectively). This free-energy landscape methodology, already applied successfully to chaperone proteins [28] and now to protein-ligand binding of human GSTA1 enzyme, is very powerful to identify network of residues and their dynamical coupling involved in the communication between domains or binding sites of biological systems. An important outcome of the present approach is its ability to decipher long-range effects, i.e., influence of residues far from the binding site on the ligand-protein association. This might guide engineered mutagenesis to tailor specific ligand-protein interactions. The following are available online at [[https://www.mdpi.com/article/10.3390/app12168196/s1](https://www.mdpi.com/article/10.3390/app12168196/s1)]([https://www.mdpi.com/article/10.3390/app12168196/s1](https://www.mdpi.com/article/10.3390/app12168196/s1)), Section S1: details about MD protocol, structural stability of hGSTA1 dimer and monomers, ligand association/dissociation, and principal component analysis applied to internal angles coordinates. Figure S1 : Identification of residues which belong to the G and H site of hGSTA1 from PDB structures. Figure S2: RMSD of hGSTA1 structures in the APO, GSH, and GS-R forms computed from MD trajectories. Figure S3: Distance between ligands and binding site G of hGSTA1 computed from MD trajectories. Figure S4: Dissimilarity index along the amino-acid sequence for side-chain angles \(\chi\). Figure S5: 2D free-energy surfaces \(V(\theta,\gamma)\) for all coarse-grained angles identified to be influenced by ligand binding in hGSTA1. Figure S6: 1D free-energy profiles \(V(\chi)\) for all side-chain dihedral angles identified to be influenced by ligand binding in hGSTA1. Figure S7: Contribution of clockwise modes extracted from PCA to the total fluctuations observed in MD. Figure S8: Cartoon structure of hGSTA1, with the network of residues relevant for ligand binding highlighted. Table S1: Summary of CGAs and SCAs involved in the ligand binding to hGSTA1 and extracted from similarity of FESs and FEPs. References [44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57] are cited in the supplementary materials. Conceptualization, A.N. and P.S.