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.