Table 1. The calculated Y548−Y470 unbinding free energies and the detailed structural statistics of WT hDAT and its mutants in the outward-occluded state.
| Mutation | WT | H547A | H547P | Y470H |
|---|---|---|---|---|
| Relative Y548-Y470 unbinding free energy (kcal/mol)a | 4.65 | 5.78 | 1.70 | 0.00 |
| Distance between the Cα atoms of Y470 and Y548 (Å)b,c | 7.37 ± 0.40 | 7.26 ± 0.39 | 8.40 ± 0.50 | 9.57 ± 0.35 |
| Cγ(D476)−Cζ(R85) distance in the D476-R85 salt bridge (Å)b,d | 4.35 ± 0.36 | 4.25 ± 0.25 | 8.31 ± 0.42 | 7.61 ± 0.62 |
| RMSD of Y470 Cα (Å)b,e | 0.58 ± 0.23 | 0.44 ± 0.18 | 0.77 ± 0.24 | 0.54 ± 0.19 |
| RMSD of R85 Cα (Å)b,e | 0.63 ± 0.19 | 0.52 ± 0.20 | 0.60 ± 0.21 | 0.53 ± 0.21 |
| RMSD of D476 Cα (Å)b,e | 0.65 ± 0.25 | 0.52 ± 0.23 | 1.27 ± 0.28 | 1.17 ± 0.28 |
aThe unbinding free energy required to separate the distance between Cα atoms of Y548 and Y470 from the lowest-energy distance to 10.25 Å (the common reference state).
bThe statistics was based on the last 5 ns of the MD simulation for each protein system.
cThe equilibrated distances between the Cα atoms of Y470 and Y548 in the outward-open, outward-occluded, and inward-open states of WT-hDAT were 7.37 ± 0.36 Å, 7.23 ± 0.35 Å, and 7.29 ± 0.37 Å, respectively. These data were based on the 50 ns MD trajectory for each system.
dThe distances between the Cγ atom of D476 and Cζ atom of R85. The equilibrated distance in the D76-R85 salt bridge in the outward-open, outward-occluded, and inward-open states were 7.76 ± 0.57 Å, 4.30 ± 0.31 Å, and 4.05 ± 0.14 Å, respectively. These data were based on the 50 ns MD trajectory.
eThe reference structure used in the RMSD calculations was the energy-minimized structure of WT hDAT in the outward-occluded state.