Structures preparation
The initial structures for molecular dynamics (MD) simulations were constructed based on the crystal structure of AChE with the PDB ID 4EY4. All buffer ions and glycerol were deleted. Missing residues were fixed by using the Galaxyfill standalone program.16 The protonation states for the titratable residues at pH 7.4 were assigned by using pdb2pqr17 and had been carefully verified by visualization. The ACh substrate was optimized by performing ab initio quantum mechanical calculations using Gaussian09 program18 at HF/6-31G* level, followed by calculations of electrostatic potentials on the molecular surfaces also at HF/6-31G* level. The calculated electrostatic potentials were used to determine partial atomic charges of ACh by using the standard restrained electrostatic potential (RESP) fitting procedure.19The missing force field parameters were taken from the general Amber force field (GAFF) implemented in Amber 18.20 Each system was neutralized by adding counter ions of 0.15 M NaCl and then was solvated in a rectangular box of TIP3P water molecules with a minimum solute wall distance of 10 Å. The number of counterions was determined by using the SLTCAP algorithm, which was supposed to more accurately capture screening effects.21 All inserted sections, including the missing residues, hydrogen atoms, waters, and counter ions, were subjected to energy-minimization followed by 1 ns conventional MD simulations to remove possible bad contacts and to equilibrate the waters and counter ions. The positional constrains with the weight of 100 kcal/(mol. Å2) were applied on the remaining atoms, i.e. , all atoms resolved in the crystal structure.
For the MD simulations of AChE-ACh complex systems, the substrate ACh was docked into the active site of AChE by using the Autodock vina program.22 Note that, to initiate the hydrolysis of ACh, the ACh carbonyl carbon atom should be close enough to the catalytic Ser203, and the carbonyl oxygen atom should be close enough to the oxyanion hole. Thus, to ensure an appropriate starting structure, we had also employed NMR constraints with the weight of 10 kcal/(mol. Å2) on the key distances between the ACh carbonyl carbon atom and the catalytic Ser203 hydroxyl oxygen atom, and on the key distances between the ACh carbonyl oxygen atom and the amide hydrogen atoms of oxyanion hole. The NMR constraints would take effect only when the distance to Ser203 was larger than 3.5 Å, or the distances to the oxyanion hole were larger than 2.5 Å.
The last snapshots of each MD simulation were taken as the initial structures for the subsequent conventional MD simulations.