Computational protocol: Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

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Protocol publication

[…] All MD simulations were performed using the GROMACS suite of programs (version 3.3) (). The AMBER99 force field (,) was employed to describe the G-riboswitch and its ligands, adenine and guanine. Partial charges of the ligands were derived using the RESP () approach, which is in line with the force field parameterization. The RNA was placed in a rhombic dodecahedron box (edge length ∼8 nm), which was subsequently filled with about 11 000 TIP3P water molecules (). To neutralize the system, 66 sodium or 33 magnesium ions were placed randomly in the simulation box. The starting structure of the guanine-bound G-riboswitch complex was taken from the Protein Data Bank (,) [PDB structure 1Y27 ()]. The G-riboswitch with adenine as the bound ligand was modeled by fitting the adenine to the ligand position in the crystallographic structure. The guanine complex and the free G-riboswitch were simulated for 150 ns, the adenine complex for 100 ns.A cut-off of 1.0 nm was used for the Lennard–Jones interactions. The interactions between atoms within 1.0 nm were evaluated at every time step. The particle mesh Ewald method () was employed to treat Coulomb interactions, using a switching distance of 1.0 nm, a grid of 0.12 nm and a beta value of 3.1 nm−1. Long-range dispersion corrections for energy and pressure were applied. Constant pressure p and temperature T were maintained by weakly coupling the system to an external bath at 1 bar and 298 K, using the Berendsen barostat and thermostat, respectively (). The system was coupled to the temperature bath with a coupling time of 0.1 ps. The pressure coupling time was 0.5 ps and the isothermal compressibility 4.5×10−5 bar−1. The bond distances and the bond angle of the solvent water were constrained using the SETTLE algorithm (). All other bond distances were constrained using the LINCS algorithm (). A leap-frog integrator with a integration time step of 2 fs was used. Analysis of the trajectories was performed with tools from the GROMACS package and with modified versions of them. To define the presence of an hydrogen bond, an acceptor–donor distance <0.35 nm and a donor-hydrogen-acceptor angle >150° was requested. Figures showing molecular structures were generated using the graphical package VMD ().To study the unbinding of the ligand, a spring (i.e. a harmonic potential) was connected to the ligand and slowly retracted (). This has the effect of pulling the ligand away from its initial location. Two pulling coordinates were used: the distance between the centers of mass of guanine and the base of residue C74 as well as the corresponding distance between guanine and U51. We employed a spring constant between 6000 and 10 000 kJ mol−1 nm−2 and a pulling rate of 0.0001 nm ps−1. For each choice of the pulling coordinate, we performed five independent 10 ns simulations of the guanine-bound G-riboswitch complex in magnesium solution (virtually the same results are obtained for sodium ions). […]

Pipeline specifications

Software tools GROMACS, P-LINCS, VMD
Application Protein structure analysis
Chemicals Adenine, Guanine