## 1 Introduction  
Proteins are biological macromolecules that perform a large variety of functions in living cells comprising biochemical (enzymes), structural, mechanical, and signaling functions. To perform their functions, proteins interact with small molecules referred to as ligands, which are able to bind to a protein with high affinity and specificity [1]. These protein/ligand interactions are crucial in biology, particularly in the context of drug design [2]. Since proteins interact with a broad range of drugs, it is of particular interest to study the mechanisms of binding of ligands to proteins and its impact on the structural dynamics to gain insights into (i) phenomena involved in the biological process and related to diseases [3] (misfolding, aggregation), and (ii) discovery, design, and development of new drugs [4]. The experimental structural data (e.g., X-ray crystallography, NMR, or cryo-EM) provide key structural information of the ligand-bound and ligand-unbound (APO) proteins [5]. Nevertheless, the static information is not always sufficient for understanding protein-ligand binding mechanisms, especially when pockets are highly flexible and contain several binding sites. Therefore, molecular dynamics (MD) is a powerful tool that provides a description of the dynamics and structures of protein-ligand systems with a high spatial and temporal resolution. Glutathione transferases (GSTs) belong to a ubiquitous superfamily of enzymes that metabolize a broad range of reactive toxic compounds by catalyzing the conjugation of reduced tripeptide glutathione (\(\gamma\)-Glu-Cys-Gly; named GSH) to the electrophilic center of a second substrate [6; 7; 8], the reactivity of GSH being due to the thiol group SH of the cysteine residue. The conjugation reaction occurs spontaneously but GST accelerates it dramatically. This process of detoxification protects cells against damages caused by both exogenous and endogenous molecules. GSTs were first discovered in liver cells [9; 10], and since then, they have been found to exhibit ligand-binding properties for a large variety of compounds, which are not always their enzymatic substrates [11]. Therefore, GSTs participate in diverse biological processes, making them multifunctional proteins. Moreover, GSTs are classified into three families according to their location in the cell: cytosolic, mitochondrial, and microsomal, which is not evolutively related to the two other classes [12]. First-discovered and most-abundant cytosolic GSTs are divided into 13 classes based on homology of their sequences. Members of the same cytosolic class have at least 40% of sequence identity, while members of different classes must have at most 25% of sequence identity. Even if they present a low homology with the cytosolic GST, mitochondrial GSTs can be considered as a particular class of GSTs (Kappa). Humans possess GST members in seven different classes [13], particularly the Alpha-class (cytosolic GSTs), in which the GSTA1 protein, which is the protein of interest in the present work, belongs. An alternative classification is possible on the basis of the residue located in the G site and which favors the activation of GSH (deprotonation of GSH plus reduction of pKa): the Cys-GSTs, whose structures are very similar to the ancestral precursor of all GSTs, the Ser-GSTs (Delta, Theta, Zeta, and Phi classes, including also Nu-GST activated by a threonine), and the Tyr-GSTs (Alpha, Pi, Mu, and Sigma classes); this latter subfamily comprises the more recently evolved GSTs. With some exceptions, GSTs are catalytically active as dimers. The GSTA1 dimer is stabilized by a \"lock and key\" motif consisting of two key residues (Met51 and Phe52) fitting into a hydrophobic cavity of the other dimer [14]. Human GSTA1 dimer revealed negative cooperativity properties depending on the substrates. It was proposed that this negative cooperativity allows the self-preservation of their functions [15; 16]. Additionally, there is no clear evidence that monomeric forms of GSTA1 are active as well as folded [17].