## 3 Results
### Structural Flexibility of Human GSTA1 in Its APO, GSH, and GS-R Form
(**Top**) panel: APO vs. GSH, (**middle**) panel: GSH vs. GS-R, and (**bottom**) panel: GS-R vs. APO. Identification of Key Residues Involved in Ligand Binding to hGSTA1 from Free-Energy Landscape Analysis of CGAs and SCAs  
The network of residues influenced by ligand binding into hGSTA1 in the three different forms presented in Figure 1C is determined by computing similarity indices \(H\) between (i) 2D free-energy surfaces of CGAs (\(\theta,\gamma\)) and (ii) 1D free-energy profiles of SCAs (\(\chi\)) along the amino-acid sequence. First, only FESs that show large or moderate similarities (\(H>0.3\)) between monomers A and B of the homodimer are considered as the _DATA_. This ensures that non-converged FESs and FEPs between the two monomers are not taken into account. In fact, even though monomers A and B can have an asymmetrical dynamical behavior from MD due to the fact they are flexible monomers (sampling issue), no information can be exploited from these non-converged characteristics from a thermodynamical point of view. They might or might not be relevant to the conformational fluctuations of the protein. In total, 11 CGAs were excluded (\(i=50\) for APO, \(i=141,145,220\) for GSH, and \(i=144,146,147,149,217,218,219,220\) for GS-R form). These CGAs are mainly located in the loop between helices \(\alpha_{5}\) and \(\alpha_{6}\) and in the C-terminal region, and they represent only 5% of the total number of CGAs (\(N=218\)). Second, we computed similarity indices \(H\) between FESs of CGAs (from _DATA_) between the different forms of hGSTA1, (i) APO vs. GSH, (ii) GSH vs. GS-R, and (iii) GS-R vs. APO. Figure 4 shows dissimilarity \(1-H\) along the amino-acid sequence for each comparison for both CGAs (\(\theta,\gamma\)) (panel A) and SCAs \(\chi^{1}\) (panel B). On one hand, from APO vs. GSH comparison, only one CGA (\(\theta,\gamma\))\({}_{i}\) presents moderate similarity of its FES between the two forms. This result is not very surprising since at the microsecond timescale, the association/dissociation of GSH ligands from G sites of monomer A and B of hGSTA1 were observed during MD simulations (see Figure S3). It concerns CGA (\(\theta,\gamma\))\({}_{143}\) and involves residues Ser142, His143, Gly144, and Gln145, which are not directly involved in the ligand-binding sites of hGSTA1. However, these residues are located at the end of \(\alpha_{5}\)-helix, which contains residue Arg131 of the G site. On the other hand, comparisons involving the GS-R form of hGSTA1 present many more structural modifications. From GSH vs. GS-R comparison, seven CGAs (\(\theta,\gamma\))\({}_{i}\) present moderate or low similarities of their FESs between these two forms, for \(i=13,14,15,16,17,18,51\). These seven CGAs involve 13 different amino acids with Gly14, Arg15, and Ser18, which are directly involved in the ligand-binding site of hGSTA1 (Figure 4C). Among the ten other residues detected and which are not located in the binding sites of hGSTA1, Arg20 is of great interest since this amino acid is highly conserved through GST classes. Moreover, by looking at FES of CGA (\(\theta,\gamma\))\({}_{15}\) in the three ligand-binding forms of hGSTA1 (Figure 4D), we show that both \(\theta\) and \(\gamma\) angles are modified by the presence of glutathione-S-conjugated ligand compared to the APO and GSH forms. For instance, the global minimum of the FES \(V(\theta_{15})\) is significantly shifted to lower values of \(\theta\) and there is the creation of a new global minimum for \(V(\gamma_{15})\) in the GS-R form compared to the GSH form. It is also the case for residues 13 and 17 (see Figure S5). Therefore, the presence of GS-R ligand modifies the structure of hGSTA1 for some residues, which corresponds to an induced fit model of protein-ligand binding. On the opposite, for the comparison between the APO and GSH forms, the population shift model of protein-ligand binding is more pronounced since the similarity between FESs and FEPs is larger than comparisons with GS-R forms (Figure 4A,B).