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.