## 3 Results  
### Structural Flexibility of Human GSTA1 in Its APO, GSH, and GS-R Form  
Figure 3A presents thermal B-factors computed during MD simulations of hGSTA1 in its APO, GSH, and GS-R form. First, the correlation between B-factors of monomers A and B in each of the three forms independently is very high (more than 80%). Therefore, we compare average B-factors computed over the two monomers for each form. Overall, four regions of the hGSTA1 enzyme were found to exhibit a large flexibility (Figure 3B): the \(\alpha_{2}\) region (residues 38-50), which contains residue Arg45 that belongs to the G site; the loop between \(\alpha_{4}\)-\(\alpha_{5}\) helices (residues 107-119), i.e., L4,5, which contains residues Leu107, Leu108, and Val111 that belong to the H site, the loop between \(\alpha_{8}\)-\(\alpha_{9}\) helices, i.e., L8,9 (residues199-206), and, finally, the C-terminal region (residues 210-222) containing the \(\alpha_{9}\) helix with residues Phe220 and Phe222 that belong to the G and H sites, respectively. By computing the difference of flexibility of hGSTA1 between the three forms shown in Figure 1C, we found that the APO and GSH forms are more flexible than the GS-R form in the \(\alpha_{2}\) region, which contains residue Arg45. This flexibility in the absence of the substrate (APO and GSH forms) may promote its binding, the same region being constrained by the presence of the conjugated substrate (GS-R form). On the opposite, the loop and extremities of \(\alpha_{4}\) and \(\alpha_{5}\) helices are more flexible in the GS-R and GSH forms of hGSTA1 than in its APO form. It means that the presence of the ligand in the G and H sites of GST and, particularly, the interactions with residues Ala100, Asp101, Ile106, Leu107, Leu108, Val111, (in \(\alpha_{4}\)) and Arg131 (in \(\alpha_{5}\)) modify the flexibility of this region (Figure 1B). Moreover, the presence of GSH in the G site strongly increases the flexibility of the loop \(L_{8,9}\) located a few residues away for the C-terminal helix \(\alpha_{9}\) of hGSTA1. This loop is also slightly flexible in the APO form and is not at all in the GS-R form. Last, but not least, the APO form of hGSTA1 shows a much larger flexibility in the C-terminal region (residue 210-222) than both the GSH and the GS-R forms. The sum of the B-factors in this region for each of the forms is 39, 21, and 12 nm\({}^{2}\) for APO, GSH, and GS-R, respectively. This result is in agreement with experimental observations showing that the protein flexibility of hGSTA1, including the dynamics of C-terminal \(\alpha_{9}\) helix on nanosecond-millisecond timescale and the protruding extremities of \(\alpha_{4}\)-\(\alpha_{5}\) helices, contributes remarkably to the catalytic and noncatalytic ligand-binding functions of GSTs [32; 33]. Figure 3: (**A**) Thermal B-factors of hGSTA1 computed from MD in its three different forms: APO (**top**), GSH (**middle**), and GS-R (**bottom**) panel. Yellow and purple circles correspond to residue that belongs to the G and H site, respectively. Gray rectangles indicate residues in \(\alpha\) helix, and black circles at the top of each plot represent residues in \(\beta\)-sheets. The Pearson correlation between monomers A and B, \(\rho_{A|B}\), is also indicated for each form. (**B**) Cartoon representation of hGSTA1 structures colored according to their B-factors in its three different forms: APO (**top**), GSH (**middle**), and GS-R (**bottom**) panel. Ligand is shown in transparent spheres, and residues which belong to the G and H sites of each monomer are shown in stick. Secondary structures of interest (see text) are also indicated: \(\alpha_{2}\)  
helix; the loop between \(\alpha_{4}\)-\(\alpha_{5}\) helices, i.e., L4,5; the loop between \(\alpha_{8}\)-\(\alpha_{9}\) helices, i.e., L8,9 and the C-terminal region (\(\alpha_{9}\) helix). The color code used is rainbow, from blue to red color corresponding to 0 and 5 nm\({}^{2}\), respectively. (**C**) Difference of B-factors between two different forms of hGSTA1.