2.7 Molecular dynamics (MD) simulations of homeodomain and its interaction
The native and mutant protein structures were subjected to MD simulations using the PMEMD program in AMBER16 package50. All the systems were solvated using an octahedral water box at an edge distance of 12 Å using a 3-point water model (TIP3P)51. Besides an ionic strength of 150mM, an appropriate number of sodium and chloride counterions were added to neutralize the charge of each system. In this study, AMBER ff14SB force field52 for protein and BSC0 for DNA 53 were used to perform MD simulations. To remove high energy contacts and steric clashes between atoms, each system was minimized by the steepest descent minimization of 2500 steps followed by a conjugate gradient minimization of 4000 steps. Then, each system was gradually heated from 0 to 298.15 K for 50 ps in NVT ensemble and equilibrated for another 600 ps with the protein-DNA position restrained using a force constant of 10 kcal/mol·Å2 and 2 kcal/mol·Å2, respectively. Simulations employed periodic boundary conditions, and long-range electrostatic interactions were estimated through Particle Mesh Ewald (PME) approaches. SHAKE was applied to bonds involving hydrogen, and the cutoff value for direct-space nonbonded interactions was set to 12.0 Å 54. The time step was set to 2 fs. The Langevin thermostat with a collision frequency of 2.0 ps−1 was adopted to regulate the temperature of the systems. The equilibration for 20 ns was carried out 298.15 K at 1 bar pressure without any restrictions and followed by a production run for 200 ns at the same conditions were used to record conformations. Root mean square fluctuation (RMSF) and hydrogen bonds were analyzed using the CPPTRAJ module of AMBER Tools55. Hydrogen bond profiles of all the simulated structures were calculated to find the number of hydrogen bonds associated with each structure. The schematic diagrams of the protein-DNA complex were deduced using DNAproDB. UCSF Chimera 1.14 56 was used to visualize protein structures and MD trajectories and prepare images. Fifteen systems including wild type-DNA and mutated IRX4-DNA systems were simulated and the interactions analysed.