3.1 Structure
Selection
and Preparation
It is essential in any MD study, to accurately select the starting
structure for simulations. In the case of the p53 DBD, through
the comparison of different PDB structures available in an MD
framework, we realized the importance of including the residues
of the N-terminal disordered extremity, as explained in the paragraphs above. In this example, it is suggested to use the X-ray
structure of the p53 DBD in the PDB entry 2XWR (chain A,
residues 91–289) [95]. This is a structure of the unbound p53
DBD including four residues from the N-terminal tail. To model
the p53 DBD DNA-bound state, we could use the PDB structure
1TSR (chains B 95-289 of p53 DBD, E and F, [109]) and carry out
a structural alignment of the two p53 DBDs, and then retain in the
PDB file for simulations only the chain A of 2XWR and remove
chain B from 1TSR. As an alternative, the missing residues could be
modeled into the 1TSR structure using 2XWR as a template. We
suggest using the chain B of 1TSR in the modelling since it is
centrally placed with respect to the DNA molecule in the crystal
structure.
To define the protonation state of the histidine, we used the
Propka server [110], whereas the cysteine and histidine residues for
Zn
2+ coordination were kept in their unprotonated state. The
N-terminus and C-terminus were also modeled at pH ¼ 7 as
positively and negatively charged moieties, respectively.
3.2 Preparatory
Steps for Classical MD
and Metadynamics
A force field has to be selected for the simulations, and we recommend to carefully read the literature with force field benchmarking
and to use those force fields that would be more suitable as a
combination to model both a protein and a DNA molecule. In
our previous publication, we tested both CHARMM22-CMAP
[111] or CHARMM22* [112] and CHARMM27 DNA parameters [113]. Nevertheless, MD force fields are continuously
adjusted or developed so that in new applications, for example, a
more recent force field could be used [114].
The systems have to be solvated in a dodecahedral box of water
molecules at 150 mM NaCl using periodic boundary conditions
with a minimum distance of at least 1.3 nm between the protein
and the box edges. In the case of the CHARMM family of force
fields, the usage of the TIP3P solvent model [115, 116] is recommended. Topology and box preparation, as well as solvation of the
system, can be carried out using the pdb2gmx, editconf, and genbox
tools from Gromacs. respectively.
Each system needs to be initially relaxed by 10,000 steps of
energy minimization with the steepest descent algorithm. The
optimization step was followed by 0.5 ns or one ns of solvent
equilibration at 300 K while restraining the protein atomic positions using a harmonic potential. Each system can be then equilibrated to the target temperature (300 K) and pressure (1 bar)
through thermalization and pressurization simulations in the
232
Elena Papaleo
Selection
and Preparation
It is essential in any MD study, to accurately select the starting
structure for simulations. In the case of the p53 DBD, through
the comparison of different PDB structures available in an MD
framework, we realized the importance of including the residues
of the N-terminal disordered extremity, as explained in the paragraphs above. In this example, it is suggested to use the X-ray
structure of the p53 DBD in the PDB entry 2XWR (chain A,
residues 91–289) [95]. This is a structure of the unbound p53
DBD including four residues from the N-terminal tail. To model
the p53 DBD DNA-bound state, we could use the PDB structure
1TSR (chains B 95-289 of p53 DBD, E and F, [109]) and carry out
a structural alignment of the two p53 DBDs, and then retain in the
PDB file for simulations only the chain A of 2XWR and remove
chain B from 1TSR. As an alternative, the missing residues could be
modeled into the 1TSR structure using 2XWR as a template. We
suggest using the chain B of 1TSR in the modelling since it is
centrally placed with respect to the DNA molecule in the crystal
structure.
To define the protonation state of the histidine, we used the
Propka server [110], whereas the cysteine and histidine residues for
Zn
2+ coordination were kept in their unprotonated state. The
N-terminus and C-terminus were also modeled at pH ¼ 7 as
positively and negatively charged moieties, respectively.
3.2 Preparatory
Steps for Classical MD
and Metadynamics
A force field has to be selected for the simulations, and we recommend to carefully read the literature with force field benchmarking
and to use those force fields that would be more suitable as a
combination to model both a protein and a DNA molecule. In
our previous publication, we tested both CHARMM22-CMAP
[111] or CHARMM22* [112] and CHARMM27 DNA parameters [113]. Nevertheless, MD force fields are continuously
adjusted or developed so that in new applications, for example, a
more recent force field could be used [114].
The systems have to be solvated in a dodecahedral box of water
molecules at 150 mM NaCl using periodic boundary conditions
with a minimum distance of at least 1.3 nm between the protein
and the box edges. In the case of the CHARMM family of force
fields, the usage of the TIP3P solvent model [115, 116] is recommended. Topology and box preparation, as well as solvation of the
system, can be carried out using the pdb2gmx, editconf, and genbox
tools from Gromacs. respectively.
Each system needs to be initially relaxed by 10,000 steps of
energy minimization with the steepest descent algorithm. The
optimization step was followed by 0.5 ns or one ns of solvent
equilibration at 300 K while restraining the protein atomic positions using a harmonic potential. Each system can be then equilibrated to the target temperature (300 K) and pressure (1 bar)
through thermalization and pressurization simulations in the
232
Elena Papaleo