) In subtractive QM-QM2 calculations, a high-level QM region interacts with a low-level QM2 region treated with the GFN-XTB/GFN2-XTB method. Since the QM2 method, i.e., GFN-XTB/GFN2-XTB, an extended semiempirical tight-binding model, is polarizable, the interaction with the high-level QM region is more accurate 100 . The GFN-XTB method accounts for anisotropic second-order density fluctuation effects, allowing the QM2 region to adjust its electron density dynamically in response to external perturbations (electron distribution) in the QM region , allowing for a more realistic description of charge redistribution and electronic polarization during interactions between the high-level QM and low-level QM2 regions. Consequently, subtractive QM-QM2 methods, compared to additive methods, improve accuracy in energy calculations, geometry optimization, and interaction energies by accurately treating electron density differences. By default, unless specified, ONIOM calculations in the ORCA engine employ the electrostatic embedding scheme, where the highlevel QM region interacts with the atomic point charges of the low-level QM2 region. In the electrostatic embedding scheme, partial charges of QM2 atoms are incorporated directly into the Hamiltonian of the QM region, i.e., electrostatic interactions between the QM and QM2 regions are explicitly considered at the QM level. These point charges are determined based on a full system low-level (QM2) calculation, ensuring that they accurately represent the charge distribution of the QM2 atoms. Different charge schemes implemented into the ORCA QM engine are available to calculate the charges of the ligand at the binding site, including the CHELPG (CHarge-Extra Loop and Gaussian Potentials), Hirshfeld, Löwdin, and Mulliken population analysis. The CHELPG algorithm was used exclusively in this study and involves fitting atomic charges to reproduce the molecular electrostatic potential at a set of grid points surrounding the molecule 102 , subject to the constraint that the sum of charges of all the atoms equals the overall charge of the molecule. Other charge analysis schemes, such as the Hirschfeld, Mulliken and Löwdin population analysis, can also be employed within the framework. The CHELPG method is chosen for reparameterizing ligand charges for the two sets of receptor-ligand complexes within the present study due to its reliability in reproducing molecular electrostatic potentials (ESP). However, as discussed previously, other charge schemes could also be employed for ligand charge parameterization at the binding site. A self-consistent field (SCF) electronic structure calculation is performed for the QM-QM2-MM system to find the electronic wave function that minimizes the electronic energy of the system, accounting for the interactions between the QM and MM regions. Once the SCF calculation converges, necessary output files, including the geometry-basis wavefunction (GBW) and the SCF electron density file, are saved. These files contain information about the molecular structure, electronic structure, and electron density distribution. The CHELPG scheme then calculates the ESP using the data from the GBW and electron density files. It performs a charge fitting procedure to assign atomic charges to each atom in the molecule to best reproduce the calculated ESP at a set of grid points surrounding the molecule. These charges represent the electron density distribution within the molecule based on the electrostatic potential. As a part of the QMrebind protocol, solvent molecules are not included in the charge calculation at the MM level. Numerous charge derivation methodologies in MD simulations exclude water or other solvent molecules , ensuring that derived charges are indicative of the inherent electronic properties of the system without being influenced by a liquid-phase, transient, high dielectric medium in its surroundings. Moreover, introducing water molecules into a charge calculation involves consideration of numerous complexities, such as selecting the number of water molecules, their respective orientations, and relative positions,