7 Experimental procedures
7.8 Molecular modeling
7.8.2 Molecular dynamics simulations
For all MD simulations, the system was simulated at 310 K and at a pressure of 1.01325 bar over 1.5 ns with recording intervals of 0.1 ps for energy and 0.5 ps for the trajectory. The OPLS 2005 Force Field [128] was used in combination with the SPC model for water [129].
The method employed for electrostatic interactions was the Particle mesh Ewald (PME) [130]
with a real space cut-off of 9.0 Å. The system was coupled to a Nose–Hoover–Chain thermostat [131] with a relaxation time of 1.0 ps and to an isotropic Martyna–Tobias–Klein barostat [132] with a relaxation time of 2.0 ps. The integration time step was set to 2.0 fs.
Data analysis was conducted using Desmond Simulation Event Analysis, and all molecular images were made with PyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC, 2011).
7.8.2.1 FUT8–GDP-Fuc complex
The system was immersed into an orthorhombic water box containing 0.2 M sodium chloride and 16,847 solvent molecules in total. The box dimensions were calculated with the buffer method in such a way that the minimal distance between the periodic images of the box was 10 Å in each dimension and the box volume was 631,032 Å2. The total number of atoms during MD simulations was 59,073.
Table 7-17: Results of the simulation quality analysis.
Block length for averaging 10.0 ps
Duration 1.5 ns
Time 1.5 ns
Degrees of freedom 120,390
Particles 16,849 (1 protein, 1 ligand, 63 sodium ions, 63 chloride ions and 16,721 water molecules)
Atoms 59,073
Target temp 310 K
Ensemble type Hoover-Noise chain thermostat, constant particle number, constant volume and constant temperature (HN_NVT)
Table 7-18: Average properties and their standard deviations of the MD simulation.
Properties Average Std. Dev. Slope (ps-1)
Total energy [kcal/mol] -158,468 9.7 0.014
Potential energy [kcal/mol] -196,039 108.6 -0.089
Temperature [K] 308.620 0.4 0.0
Pressure [bar] -1107 14.1 0.002
Volume [A³] 631,032 0.0 0.0
Figure 7-1: Behavior of properties during the MD simulation of the FUT8–GDP-Fuc complex over time.
0 200 400 600 800 1000 1200 1400 1600
-1.5844e5 0
-20 -40 Total Energy [kcal/mol] -60
Time Step [ps]
Pot. Energy [kcal/mol]
0
-800 -600 -400 -200
-1.956e5
Pressure [bar]
-1150 -1130 -1110 -1090 -1070
Temperature [K]
310 309 308
Volume [A³]
600000 620000 640000 660000
Figure 7-2: Root mean square deviation (RMSD, black) and Root mean square fluctuation (RMSF, purple) of all residues in the MD simulation of the FUT8–GDP-Fuc complex.
7.8.2.2 FUT8 ternary complex
MD simulations are based on the model the complex of FUT8 and GDP-Fuc [108]. As a starting point, last frame of the MD simulation of the FUT8–GDP-Fuc complex was used. A model of the heptasaccharide 1b was manually docked into the putative binding pocket as described in Section 4.3.3. After minimization of the ternary complex, the system was immersed into an orthorhombic water box containing 0.2 M sodium chloride and 58,697 solvent molecules in total. The box dimensions were calculated with the buffer method in such a way that the minimal distance between the periodic images of the box was 10 Å in each dimension and the box volume was 713,764 Å2. The total number of atoms during MD simulations was 66,429. The MD simulation was performed with the parameters described above.
Table 7-19: Results of the simulation quality analysis for the MD of the ternary complex.
Block length for averaging 10.0 ps
Duration 1.5 ns
Time 1.5 ns
Degrees of freedom 136,929
Particles 58,700 (1 protein, 2 ligands, 76 sodium ions, 76 chloride ions and 58,545 water molecules)
Atoms 66,429
Target temp 310 K
-200 0 200 400 600 800 1000 1200 1400 1600 0
2 4 6 8
[Å]
Time Step [ps]
RMSD RMSF
Table 7-20: Average properties and their standard deviations of the MD simulation.
Properties Average Std. Dev. Slope (ps-1)
Total energy (kcal/mol) -18,1387 57 0.13
Potential energy (kcal/mol) -224,260 112 -0.044
Temperature (K) 308.65 0.4 0.0
Pressure (bar) -959.8 14.3 0.0
Volume (A³) 713,764.01 0.0 0.0
Figure 7-3: Behavior of properties during the MD simulation of the ternary complex over time.
0 200 400 600 800 1000 1200 1400 1600
-1.812e5 -50 -150 -200 Total Energy [kcal/mol]
Time Step [ps]
Pot. Energy [kcal/mol]
0
-600 -400 -200
-2.24e5
Pressure [bar]
-1000 -980 -960 -940 -910
Temperature [K]
310 309 308
Volume [A³]
680000 700000 720000 740000 -300
307
Figure 7-4: Root mean square deviation (RMSD, black) and Root mean square fluctuation (RMSF, purple) of all heavy atoms in the MD simulation. As the potential energy is stable throughout the simulation (cf. Figure 7-3), the higher RMSF and the increase in RMSD are explained by rearrangement of water molecules during the first 200 ps.
Figure 7-5: Trajectories of distances of atoms of FUT8 to atoms of the GlcNAc-1 of the acceptor substrate.
-200 0 200 400 600 800 1000 1200 1400 1600 0
2 4 6 8 10
Deviation [Å]
Time Step [ps]
RMSD (heavy atoms) RMSF (heavy atoms)
Figure 7-6: Trajectories of distances of atoms of FUT8 to atoms of the GlcNAc-2 of the acceptor substrate.
Figure 7-7: Trajectory of distances of atoms of FUT8 to atoms of the Man-3 of the acceptor substrate.
Figure 7-8: Trajectories of distances of atoms of FUT8 to atoms of the Man-4 of the acceptor substrate.
Figure 7-9: Trajectories of distances of atoms of FUT8 to atoms of the GlcNAc5 (3-mannose branch) of the acceptor substrate.
Figure 7-10: Trajectories of distances of atoms of FUT8 to the atoms of Man-4' (6-mannose branch) of the acceptor substrate.
Figure 7-11: Trajectories of distances of atoms of FUT8 to the atoms (oxygen-3 and nitrogen) of GlcNAc5' (6-mannose branch) of the acceptor substrate.
Figure 7-12: Trajectories of distances of atoms of FUT8 to the oxygen-4 atom of GlcNAc5' (6-mannose branch) of the acceptor substrate.
Figure 7-13: Trajectories of distances of atoms the donor substrate and of the acceptor substrate.
7.8.2.3 FUT8 ternary complex with chitotriose as acceptor substrate
MD simulations are based on the model the complex of FUT8 and GDP-Fuc [108]. As a starting point, last frame of the MD simulation of the FUT8 ternary complex was used. A model of the 1--N-Actetyl chitotriose was manually docked into the putative binding pocket by means of fitting to the core trisaccharide of the heptasaccharide. The heptasaccharide was subsequently deleted and the system was minimized. The system was immersed into an orthorhombic water box containing 0.2 M sodium chloride and 51,862 solvent molecules in total. The box dimensions were calculated with the buffer method in such a way that the minimal distance between the periodic images of the box was 10 Å in each dimension and the box volume was 639,787 Å2. The total number of atoms during MD simulations was 59,504.
The MD simulation was performed with the parameters described above.
Table 7-21: Results of the simulation quality analysis for the MD of the ternary complex with chitotriose.
Block length for averaging 10.0 ps
Duration 1.5 ns
Time 1.5 ns
Degrees of freedom 123,016
Particles 51,865 (1 protein, 2 ligands, 68 sodium ions, 68 chloride ions and 51,726 water molecules)
Atoms 59,504
Target temp 310 K
Ensemble type Hoover-Noise chain thermostat, constant particle number, constant volume and constant temperature (HN_NVT)
Table 7-22: Average properties and their standard deviations of the MD simulation.
Properties Average Std. Dev. Slope (ps-1)
Total energy (kcal/mol) -162,197 31 0.068
Potential energy (kcal/mol) -200,723 126 -0.14
Temperature (K) 308.62 0.44 0.0
Pressure (bar) -1000 16 0.0
Volume (A³) 639,786.58 0.0 0.0
Figure 7-14: Behavior of properties during the MD simulation over time.
Figure 7-15: Root mean square deviation (RMSD, black) and Root mean square fluctuation (RMSF, purple) of all heavy atoms in the MD simulation. As the potential energy is stable throughout the simulation (cf. Figure 7-3), the higher RMSF and the increase in RMSD are explained by rearrangement of water molecules during the first 200 ps.
0 200 400 600 800 1000 1200 1400 1600
-1.621e5 -40 -80 -120 Total Energy [kcal/mol]
Time Step [ps]
Pot. Energy [kcal/mol]
0
-800 -600 -200
Pressure [bar]
-1040 -1020 -1000 -980 -960
Temperature [K]
310 309 308
Volume [A³]
600000 620000 640000 660000 -160
307 -400
680000 -2.003e5
-200 0 200 400 600 800 1000 1200 1400 1600
0 1 2 3 4 5 6
Deviation [Å]
RMSD (heavy atoms) RMSF (heavy atoms)
Time Step [ps]