## 3 Results
### Structural Flexibility of Human GSTA1 in Its APO, GSH, and GS-R Form
Finally, 10 FESs of CGAs were identified as moderately or largely modified due to ligand binding for the comparison between GS-R and APO forms of hGSTA1. In detail, CGAs (\(\theta,\gamma\))\({}_{i}\) for \(i=10,12,13,14,15,16,17,18,53\), and 158 were identified. Among these 10 CGAs, six were already detected from GSH vs. GS-R comparison (Table S1). It means that these six CGAs are GS-R specific, i.e., they are similar in the APO and GSH forms compared to the GS-R form. Particularly, CGA (\(\theta,\gamma\))\({}_{53}\), which involves residues Gln54 and Val55, is of special interest since both residues belong to the G site. In addition, CGA \((\theta,\gamma)_{158}\), which involves residue His159, is also of special interest since this residue belongs to the H site. In total, from the three different comparisons of ligand-binding forms of hGSTA1 shown in Figure 4A, 12 unique CGAs were identified to be influenced by the absence/presence of a ligand; some of them being detected in two different comparisons (Table S1). It corresponds to a total of 26 different residues with four residues which belong to the G site (Tyr9, Arg15, Gln54, and Val55) and four residues which belong to the H site (Phe10, Gly14, Ser18, and His159). All 2D FESs of the 12 CGAs identified to be involved in the ligand binding process in hGSTA1 are presented in Figure S5. We applied the exact same procedure to compare the one-dimensional FEPs of SCAs \(\chi^{k}\). First, three amino acids were excluded from the _DATA_ since they do not exhibit converged  
Figure 4: (**A**) Dissimilarity index \(1-H\) along the amino-acid sequence computed from FESs of CGAs \((\theta,\gamma)\). The color code is the following: low dissimilarity: blue, moderate dissimilarity: green, large dissimilarity: red (see Materials and Methods, Section 2.2). Yellow and purple circles correspond to residue that belongs to the G and H site, respectively. Gray rectangles indicate residues in a helix, and black circles at the top of each plot represent residues in \(\beta\)-sheets. (**B**) Dissimilarity index \(1-H\) along the amino-acid sequence computed from FEPs of SCAs \(\chi^{1}\). The color code is the same as in panel A. (**C**) Location of CGAs and SCAs detected from free-energy landscape analysis in the hGSTA1 structure. The color code is the following: N-term (blue) to C-term (red). (**D**) Effective 2D FESs \(V(\theta,\gamma)_{15}\) in the APO, GSH, and GS-R forms of hGSTA1. Effective 1D FEPs of each internal coordinate \(\theta_{15}\) and \(\gamma_{15}\) are also presented. (**E**) Effective FEPs \(V(\chi^{3})_{15}\) in the APO, GSH, and GS-R forms of hGSTA1. Diamonds indicate the global minimum in each form. FEPs of SCAs between monomers A and B of hGSTA1. It concerns residues Glu146 (\(\chi^{1,2}\)), His159(\(\chi^{1,2}\)), and Arg221 (\(\chi^{1,2,3,4,5}\)). It represents only 1.5% of the total number of residues characterized by SCAs (\(N=192\)). Then, from dissimilarity indices, \(1-H\), computed along the amino-acid sequence for the three different comparisons APO vs. GSH, GSH vs. GS-R, and GS-R vs. APO, as shown in Figure 4B for SCAs \(\chi^{1}\) and in Figure S3 for the other SCAs \(\chi^{2,3,4,5}\), a total of 19 SCAs were identified to be influenced by the absence/presence of a ligand in the binding sites of hGSTA1 homodimer. It concerns 15 different residues, i.e., Phe10, Arg13, Arg15, Met16, Glu17, Thr19, Arg45, Gln54, Leu102, Phe136, His143, Arg155, Leu163, Phe220, and Phe222 (Table S1). Among these 15 residues, four are located in the G site (Arg15, Arg45, Gln54, and Phe220) and two in the H site (Phe10 and Phe222). In detail, the comparison between the APO and GSH forms of hGSTA1 highlights three SCAs, \(\chi^{12}_{143}\) and \(\chi^{1}_{222}\) (C-term). Particularly, residue His143 was also found to be crucial (the only one) for ligand binding from the analysis of the CGA (\(\theta_{i},\gamma_{i}\)) FESs.