2. Methodology
To model the all-atom structure of Nap1-nonP, an experimentally
determined structure of yNap1 (PDB ID: 2Z2R)13 was
solvated with the TIP3P water model14 in a rectangular
box. The dimeric yNap1 molecule consists of 417 residues, of which
residues 74–365 were used for the modeling of Nap1-nonP. The
experimentally undetermined loop regions were modeled with a homology
modeling tool (Phyre2),1515 with the N- and C-terminal
coils omitted to reduce the system size. Additionally, because each
nuclear localization sequence (NLS) region contains a long β-sheet
protruding from the protein surface, the size of the periodic boundary
box would need to be increased when these regions are modeled
explicitly. Therefore, to reduce the system size, we omitted the NLS
regions by replacing them with Gly, Gly, and Ser. Figure 1 shows the
modeled all-atom structure of Nap1-nonP.
To address the phosphorylation effect on the accessory domain, an
all-atom system of Nap1-P was also prepared. To model Nap1-P, Nap1-nonP
was phosphorylated using PyTMs (a PyMol plugin) by employing a set of
phosphoserine parameters proposed in a previous
study.16 The Ser 140, Ser 159, and Ser 177 residues
were considered as the set of phosphorylation sites. Finally, the total
number of atoms, including the water molecules, was 894,611 for the
Nap1-nonP system and 894,527 for the Nap1-P system.
To extend the MD simulation time step to 2.0 fs, we constrained the
chemical bonds of the proteins and water molecules with the
LINCS1717 and the SETTLE
algorithms,18 respectively. The temperature and
pressure of the systems were controlled using the modified Berendsen
thermostat1919 and the Parrinello–Rahman
method,20,21 respectively. The electrostatic
interactions were evaluated with the particle mesh Ewald
method22 using a real-space cutoff of 10.0 Å. The
cutoff value for van der Waals interactions was set to 10.0 Å. All MD
simulations were performed with the GPU version of the GROMACS 2019
package,23 using the AMBER 14SBonlysc force
field.24 To equilibrate each solvated system, 10 ps of
the NVT ensemble (T = 300 K) and 10 ps of the NPTensemble (T = 300 K and P = 1 bar) were performed
sequentially after short energy minimization, where the last snapshots
of the NPT simulations were employed as the starting structures
of the production runs. Finally, five trials of 100-ns MD simulations
were started from the relaxed structures of both systems to obtain
statistically reliable trajectories.