2.7 Molecular dynamic simulation
Human interleukin-18 (IL-18) crystal structure was taken from RCSB
Protein Data Bank, an ascension code 3WO2 (Tsutsumi et al., 2014). The
co-crystallized solvents were then removed from the structure. The
cysteine-serine replacement was performed using FoldX package
(Schymkowitz et al., 2005). All ionizable amino acids was set as a
default state at pH 7.0. A neutral histidine was set as a singly
Ɛ-protonated histidine (HIE in AMBER name). No doubly protonated
histidine and disulfide linkage was observed. The IL18 protein was then
solvated using the TIP3P water rectangular box with the distance of 14 Å
from protein surface. The system was neutralized using sodium ion, and
sodium chloride (NaCl) was added to create 0.15 mol L-1 NaCl solution
(Saetang et al., 2016) using AMBER20 force field (Case et al., 2021) via
Leap module in AMBER20 package. In total, the system consisted of
monomeric IL18, 4 sodium ions, 33 NaCl pairs, and 11437 TIP3P waters.
The wild-type and mutated IL18 structures were then energy-minimized
using steeping descent and conjugate gradient methods for 1000 and 1000
steps, respectively under a periodic boundary condition. The minimized
structure was taken into canonical (NVT) simulation at 37oC (310 K) in which all protein positions were
restrained by harmonic potential. The temperature was controlled using
Langevin thermostat. All nonbonded and electrostatic interactions were
calculated using a cutoff of 16 Å. Each NVT restrained simulation was
executed for 200 picoseconds (ps) with a time step of 1 femtoseconds,
which a force constant of 200, 100, 50, 20 and 10 kcal/mol Å-2 , in
total of 1 nanosecond (ns). To apply the pressure of 1.013 bar (1 atm),
the system was changed into isobaric-isothermal (NPT) simulation. The
temperature and the pressure were regulated using weak-coupling
algorithm (Berendsen et al., 1984). The NPT simulation lasted 120 ns
with a time step of 2 fs. The first 60 ns simulation was omitted and the
last 60 ns simulation was taken into the analyzed trajectory. All energy
minimization and molecular dynamics processes were carried out with
PMEMD module implemented in AMBER20 package. In addition, the trajectory
in AMBER coordinate format (.mdcrd) file was converted into the Protein
Data Bank (PDB) file using cpptraj in AMBER20 package. The structure
visualization and interaction analysis of all IL18 cases were all
performed using VMD package (Humphrey et al., 1996).