Molecular Dynamics (MD) simulation of RpoE10 and its mutants
To provide atomistic details of bimolecular motions and an additional
perspective relative to experimental results, we performed
“computational microscopy” using molecular dynamics (MD) simulations
for 200 ns for each of structural models of (i) RpoE10 native; (ii)
RpoE10(Mut1) (in silico substitution of NPDKV with NAAAV) and
(iii) RpoE10(Mut2) (in silico substitution of DGGGR to AAAGR)
(iv) RpoE10 (Del1). All simulations were performed with the GROMACS
2019.2 simulation suite (http://www.gromacs.org/) using the CHARMM 27
force field parameter set 23-25. In the first step of
MD simulation, the selected homology model of RpoE10, native, and the
two mutants, Mut1 and Mut2, were solvated in a cubic water box. The
system was solvated with TIP3P water molecules. Periodic boundary
conditions were applied in all directions, and
Na+Cl− counter ions were added to
make the system electrically neutral. Then, the systems’ energy
minimization was done by 50,000 steps of steepest descents to relax any
steric conflicts. NVT and NPT equilibration then followed energy
minimization for 300ps. Once equilibrated regarding pressure and
density, these systems were then subjected to MD simulation at 300K for
200 ns, with no restrictions on the residues. During the simulation, the
temperature of the system was maintained at 300K using a Berendsen
thermostat. The particle mesh Ewald method 26 was
employed to account for the long-range electrostatic interactions, and
the LINCS algorithm 27 was used, with a time step of 2
fs to restrain bond lengths. The constant temperature and pressure (300K
and 1bar) were maintained using a V-rescale thermostat28 and Parrinello-Rahmanbarostat 29.
The production run was performed for 200ns with leapfrog integrator30, and the coordinates were saved every
10picoseconds. A total of 20,000 frames were generated. MD trajectories
were analyzed using the tools of GROMACS 2019.2. The backbone
root-mean-square deviations (RMSDs) of the RpoE10, RpoE10 (Mut-1 and
Mut-2), and RpoE10 (Del1) derivatives were calculated following the
structural alignment of the core structure. The RMSD and RMSF values
depict the regions that fluctuate differentially regarding other regions
of the protein of interest. The GROMACS analysis plots were generated by
XMGRACE utility from Grace software
(http://plasma-gate.weizmann.ac.il/Grace/). UCFC Chimera22 and VMD 31 software were employed
to analyze and visualize the molecular interactions and MD trajectories
to create molecular graphics.