Substrate binding and inhibition mechanism of norepinephrine transporter

MD simulations was performed to validate the stability of the docking pose of radafaxine. The solvated MD system of NET complexed with radafaxine was constructed. Protonation states of residues were assigned as predicted by H++. All of the disulfide bonds in the structure were preserved. Then, the complex was inserted into 100 × 74 Å2 bilayers. The system was solvated in a TIP3P water box and neutralized with 0.15 M NaCl. The dimension of system was 100 × 74 × 100 Å3. The AMBER ff14SB and GAFF2 force field was used to simulate the protein and the small molecule, respectively. For the solvated system, a 5000-step energy minimization was performed to remove conflicts and overlaps between atoms. Then, the complex was equilibrated for 500 ps in a constant-volume ensemble to heat the system from 0 to 300 K, followed for 500 ps in a constant-pressure ensemble, specifically at 1 bar. During equilibration, a force constant of 20 kcal·mol−1·Å−2 as a harmonic constraint was applied to the complex. With the constraint released, a 100-ns MD simulation was performed in constant-pressure ensembles at 300 K with 2-fs timestep. The cut-off value of the van der Waals interactions was set to 10 Å. The particle mesh Ewald (PME) method was used to calculate the long-range electrostatic contributions. The SHAKE algorithm was used to restrain all of the bond lengths involving hydrogen atoms. The AMBER20 software package was employed to carry out MD simulation. After simulation, the cpptraj module of AmberTools20 was used to calculate root mean square deviations (RMSDs) of receptor and ligand, and the top clustered structure of the complex based RMSD calculation.