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).