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.