## 4 Discussion  
From the three different comparisons of ligand-binding forms of human GSTA1 shown in Figure 1C, 12 CGAs and 19 SCAs were identified to be influenced by the absence/presence of ligands, most of them being glutathione-S-conjugated-specific (Table S1). It corresponds to a total of 33 different amino acids (15% of the total sequence), eight of them being identified from both CGAs and SCAs analyses (Table 1). Among these 33 residues, 11 of them (32%) are directly located to the two binding sites of hGSTA1, with 6 which belong to the G site, i.e., Tyr9, Arg15, Arg45, Gln54, Val55, Phe220, and 5 which belong to the H site, i.e., Phe10, Gly14, Ser18, His159, and Phe222 (Figure S8). Furthermore, a collective motion involving residues Arg15, Gln54, and Val55 of the G site and residue Gly14 of the H site was identified using principal component analysis, revealing a strong coupling between these residues. Particularly, residue Arg15, which is found here to be involved in both local conformational changes of the main chain and of the side chains of hGSTA1, was demonstrated to have crucial catalytic activity both theoretically [35] and experimentally [36; 37]. Finally, residues Phe220 (G site) and Phe222 (H site) belong to the C-terminal part of hGSTA1 (\(\alpha_{9}\)-helix; see Figure S8) and it has been demonstrated that this secondary structure contributes remarkably to the catalytic and noncatalytic ligand-binding functions of GSTs [32; 33]. At large distance from the ligand-binding sites of hGSTA1, 12 residues (from Leu102 to Leu163; see Table 1) were identified as sensitive to the ligand disturbance. For example, His143 is involved in the main-chain and side-chain conformational changes of hGSTA1 and is located around 30 A away from the G and H sites Figure S8). Moreover, GST sequence analysis indicates that some residues form an highly conserved local sequence GXXh(T/S)XDMh (h: hydrophobic), constituted by the \(\alpha_{6}\)-helix and its preceding loop [38; 39; 40]. This motif belongs to a substructure named N-capping box and hydrophobic staple motif of GST and has been shown to be critical for the protein folding and stability [41]. In the present work, six residues were identified from the \(\alpha_{6}\)-helix, particularly Arg155, Asp157 (highly conserved), and Ile158 (hydrophobic) which belongs to the corresponding motif GNKLSRADI. According to experimental structures, Ile158 can form hydrophobic interactions with the \(\alpha_{1}\)-helix [41], which is an important structural element supporting the active binding site of GSTs. Finally, it has been reported that the sequence alignment of all known GST structures (more than 100) shows that only 6-7 residues, i.e., less than 5% of the entire polypeptide chain, are strictly conserved [42]. For instance, Pro56 of the GST Alpha class, which belongs to the G site, is conserved through GST classes Mu, Pi, Sigma, Phi, Tau, Theta, Zeta, Omega, and Beta. In the present work, four amino acids, i.e., Arg13, Arg20, Val55, and Asp157, were detected and are highly conserved among GST classes. Particularly, Arg20 is strictly conserved through GST classes Alpha, Mu, Pi, Sigma, Tau, Zeta, and Omega; Arg13 is strictly conserved through GST classes Alpha, Mu, and Pi [43]. \begin{table}
\begin{tabular}{c c} \hline \hline
**Location** & **List of Amino Acids** \\ \hline G site & Tyr9 (\(\beta_{1}\)), Arg15 (L\({}_{\beta_{1},\alpha_{1}}\)), Arg45 (\(\alpha_{2}\)), Gln54, Val55 (L\({}_{\alpha_{1},\beta_{5}}\)), Phe220 (\(\alpha_{9}\)) \\ \hline H site & Phe10, Gly14 (L\({}_{\beta_{1},\alpha_{1}}\)), Ser18 (\(\alpha_{1}\)), His159 (\(\alpha_{6}\)), Phe222 (\(\alpha_{9}\)) \\ \hline \multirow{3}{*}{Others} & Asn11, Ala12, **Arg13** (L\({}_{\beta_{1},\alpha_{1}}\)), Met16, Glu17, Thr19, **Arg20** (\(\alpha_{1}\)), Leu50, Met51, \\  & Phe52, Gln53 (\(\overline{\text{L}}_{\alpha_{2},\beta_{5}}\)), Leu102 (\(\alpha_{4}\)), Phe136, Ser142, His143, Gly144, Gln145 (\(\alpha_{5}\)), Arg155, **Asp157**, Ile158, Leu160, Leu163 (\(\alpha_{6}\)) \\ \hline \hline \end{tabular}
\end{table}