Figure 1.
The system simulated is a hSERT dimer embedded in a POPC bilayer solvated with TIP3P [52] water and neutralized with NaCl to a concentration of 0.2 M. The protomers in the dimer are shown in green and blue cartoons, whereas the lipid bilayer is shown in spheres. Water is shown as a transparent surface. The approximate dimensions of the simulation system are included.
Figure 2.
Gate interactions and intracellular pathway.
Transition to the inward-facing conformation causes disruption of the intracellular gate but not the extracellular gate, and solvation of the cytoplasmic pathway to the substrate site occurs. A. The extracellular gate interactions remain stable throughout simulation as illustrated by the depicted shortest distance between the carboxylate oxygen atoms of Glu493 and guanidinium nitrogen atoms of Arg104. B. Arg104 and Glu493 form an ionic interaction and contribute to the extracellular gate. Tyr176 and Phe335 form an aromatic lid on top of the central binding pocket lined by TM1 (red), TM3 (blue), TM6 (green), and TM8 (yellow). Protein side chains are shown in gray, and the 5-HT substrate is colored by atom type with carbons in purple. C. The shortest distance between Asp452 carboxylate oxygen atoms and Arg79 guadinium nitrogen atoms in the intracellular gate are seen. D. In the intracellular gate, Arg79 and Asp452 form an ionic interaction, and Tyr350 and Glu444 interacts via a hydrogen bond. The N-terminal is shown in red, TM6 in green and TM8 in yellow. Protein side chains are shown in gray sticks. E. Calculated SASAs of the proposed cytoplasmic pathway residues (Phe88, Ser91, Gly94, Gly273, Ser277, Val281, Thr284, Phe347, Ala441, Glu444, and Thr448) are shown. The SASA of Sim8 (light blue line) increases from around 200 Å2 to 400 Å2 after 35 ns of simulation. F. Position of cytoplasmic pathway residues in hSERT. TM1, TM5, TM6 and TM8 are shown in light salmon. The remaining TMs are shown in beige. The residues found experimentally to line the cytoplasmic pathway [24], [32]–[34] are displayed in sticks surrounded by transparent surfaces and colored with those from TM1 in red, TM5 in orange, TM6 in green and TM8 in yellow. The POPC bilayer is shown in spheres and colored by atom type with light blue carbons.
Figure 3.
Comparison of solvation from the extracellular and intracellular sides.
In Sim8 the transition to the inward-facing conformation causes the intracellular pathway and substrate to be solvated by water from the cytoplasm. Solvation patterns of the ligand are shown as the number of water molecules within 3 Å (dark green) and 4 Å (light green) from the extracellular (top) and intracellular cavities (bottom) in system Sim3 (A) and system Sim8 (B). Number of water molecules located in the extracellular (EC, dark blue, top) and the intracellular side (IC, light blue, bottom) in system Sim3 (C) and system Sim8 (D). Data are extracted from 1000 snapshots during the trajectories.
Figure 4.
Na2 coordination and solvation.
Na2-ion coordination is less stringent upon transition to the inward-facing conformation. A. Traces represent distances in Sim8 between the Na2-ion and the initial coordination residues in the Na2-site. The number of residues coordinating the Na2-ion decreases during the simulation with Asp437 and Gly94 being the only continuous coordination partners. B. The Na2-ion becomes solvated upon transition to the inward-facing conformation. Dots represent the number of water molecules interacting with the Na2-ion in Sim8. After a few ns the number of water molecules within 3 Å increase to 4–5, which then substitute for the coordination from the protein.
Figure 5.
The transition to the inward-facing conformation opens up a pathway towards the cytoplasm for the Na2-ion lined by TM1 (red), TM5 (orange), TM6 (green), and TM8 (yellow). The 5-HT substrate is shown in spheres and colored by atom type, with carbons in purple. Protein residues are shown in gray sticks, and the Na2-ion is shown as a cyan sphere with the initial position of the Na2-ion indicated as a transparent light blue sphere. Water molecules found in the intracellular cavity is indicated by light gray sticks and a transparent surface. A. The snapshot after 33.9 ns molecular dynamics simulation in Sim8 where the Na2-ion is located just below the initial binding pocket formed by Ser438, Leu434, Val97, Gly94, and Asp437. The only coordination partners remaining are Asp437 and Gly94, which are located in the bottom of the ion binding site. B. Molecular dynamics snapshot after 75.4 ns (33.9 ns snapshot from Sim8 and additional 41.5 ns in Sim8b). The Na2-ion is partly transported and located between the two aromatic residues Phe347 and Phe440 and coordinated by the acidic residue Glu444. C. The Na2-ion is completely transported after 80.4 ns (33.9 ns in Sim8 and additional 46.5 ns in Sim8b) and is fully solvated by water and does not possess any interactions with the protein. D. The monitored positions of the Na2-ion in Sim8b are displayed as cyan spheres during the additional 50 ns of simulation started from the Sim8 snapshot. The spheres indicate the transport pathway of the Na2-ion from the central ion binding site to the cytoplasm. It can be observed that TM1, TM5, TM6, and TM8 are lining the transport pathway.
Figure 6.
Effects of Asp437Asn mutation.
Mutation of Asp437 to Asn in the Na2-site severely compromises the apparent transport affinity for sodium. Radiotracer uptake of 10 µM 5-HT into HEK293MSR cells transiently transfected with hSERT wt or the Asp437Asn mutant reveals that removal of the sodium coordinating side chain of residue 437 does not affect maximum transport rates negatively whereas sodium affinity is significantly impaired. NaCl is substituted with N-methyl-D-glucamine. Data is fitted to Michaelis-Menten kinetics.
Table 1.
Kinetic parameters from radiotracer uptake experiments.
Figure 7.
Comparison of pores in outward, occluded and inward-facing hSERT.
The pore running through the transporter is found to open dramatically towards the cytoplasm during the simulations reflecting an inward-facing transporter. The pore diameter is measured utilizing the HOLE plug-in for VMD [44], [45]. A. Comparison of the pore diameter through the protein is shown for the inward-facing 5-HT/ions molecular dynamics snapshot at 33.9 ns in Sim8 (cyan), the hSERT homology model (purple), an outward-facing apo/ions hSERT molecular dynamics snapshot from Sim26 (yellow), open-occluded LeuT (PDB:2A65, light green) [8], tryptophan bound outward-facing LeuT (PDB:3F3A, dark blue) [9], inward-facing LeuT by Forrest et al. (dark green) [24] and inward-facing LeuT by Shaikh and Tajkhorshid (red) [42]. B–D. Molecular illustrations of the pore running through hSERT as found in Sim26 of the apo/ions simulation after 74 ns (B), in the hSERT 5-HT/ions homology model (C), and in the hSERT 5-HT/ions molecular dynamics snapshot at 33.9 ns in Sim8 (D). The pore is shown in light blue surface in B–D. TM1, TM5, TM6, TM8, and TM10 are colored in red, orange, green, yellow, and ice blue respectively, while TM2, TM3, TM4, TM7, and TM9 are shown in gray. The residues that have been shown to line the cytoplasmic pathway of hSERT from substituted cysteine accessibility scanning [24], [32]–[34] are shown in spheres using the colors corresponding to the TM they belong to. All residues experimentally shown to increase accessibility upon inward-facing conformation are included. The residues involved in the intracellular gate (Arg79, Val274, Tyr350, Asp452 and Glu444) are shown in sticks using the same color as the TMs they belong to.
Figure 8.
Helix movements leading to conformational changes.
The intracellular parts of TM1 and TM6 are the most flexible helices in the transition to the inward-facing conformation. TM helix movements in the Sim8 system are represented by the relative movement compared to the scaffold and the starting structure. The simulations disclose a partly un-coupled movement of the two halves of the unwound helices, TM1 and TM6. A. The angles between scaffold/bundle (dark blue), scaffold/TM1a (dark red), scaffold/TM1b (red), scaffold/TM6b (dark green), scaffold/TM6b (light-green), scaffold/TM2 (cyan), scaffold/TM7 (dark-orange) are depicted along the trajectory in Sim8. B) The angles between TM1a/TM1b (yellow), TM6a/TM6b (orange) and the protein features between which the helix-tilts are measured are shown schematically in colors corresponding to the lines in the graphs. C. The movements of the different protein parts between the starting structure of Sim8 and the snapshot after 33.9 ns within the scaffold (left) and the bundle (right) are depicted. The intracellular part of the four-helix bundle (red and green helices) is moving the most. The movement is illustrated based on a structural alignment on TM3 and TM8 forming part of the hash-motif [42].
Figure 9.
Conformational states and sampling of the four-helix bundle.
The hSERT structures adapt conformational stages comparable with experimentally known outward-occluded and inward-facing states. During the trajectory in Sim8 various conformational stages are sampled, with the largest movement occurring on the intracellular side of the four-helix bundle. A. The scaffold is illustrated by light gray surface with the last ten residues of TM10 omitted for clarity. The vectors representing the axis of the four-helix bundle are shown by colored lines. The four-helix bundle of the inward-facing conformation of hSERT obtained from the 33.9 ns snapshot in Sim8 and the reference homology model are shown in ribbons colored in cyan and purple, respectively. The location of the four-helix bundle of the reference homology model (purple) and the outward-facing conformation of hSERT from the apo/ions Sim26 74 ns snapshot (orange) are very similar to what was observed from the crystal structure of LeuT with leucine bound (not shown as it overlays perfectly with the reference homology model in purple). The outward-facing conformation of LeuT with tryptophan bound (dark blue) has the vector of the four-helix bundle further displaced from the extracellular surface of the scaffold than any of the other structures. The inward-facing conformation of hSERT from Sim8 (cyan) is as open to the intracellular side as the inward-facing conformation of vSGLT (red) in the crystal structure. B. The scaffold is illustrated similar to in (A) and for clarity the bundle is removed. The vectors corresponding to arrangements of the four-helix bundle during the entire trajectory of Sim8 are illustrated using snapshots collected every 10 ns starting with the black vector changing from green to blue. The four-helix bundle sample different conformations during the simulation. The arrangement of the four-helix bundle starts from a conformation similar to the outward-occluded LeuT structure and reaches an arrangement similar to the conformation seen in inward-facing vSGLT. The largest movements of the four-helix bundle are seen to occur at the intracellular side.
Figure 10.
Overview of hSERT conformations and the binding of noribogaine to the inward-facing state.
A. The three major conformations and the conformational transitions in 5-HT transport by hSERT is shown with cut-through sections of the transporter viewed from the plane of the membrane bilayer. Left: Outward-facing apo/ions molecular dynamics snapshot (blue) from Sim26, Middle: 5-HT/ions homology model in an occluded state (green), Right: Inward-facing conformation of hSERT from Sim8 at 33.9 ns of simulation (cyan). These three structures reveal the principal states occurring during transport in hSERT. B. Bottom view of the transporter, which is illustrated by surfaces. TM1 (red), TM5 (orange) TM6 (green) and TM8 (yellow) known to line the cytoplasmic pathway have been highlighted together with the ligand in pink. In hSERT homology models which are outward-occluded, no cytoplasmic pathway is found, and the ligand cannot be seen from the cytoplasmic side. C. The snapshot from Sim8 displaying an inward-facing conformation. A direct connection to the ligand from the intracellular space is found. Only TM1–TM10 are shown here for clarity and comparison. D. Docking pose of noribogaine (cyan) in the inward facing snapshot of hSERT from Sim8 is seen. The ligand is located in the central binding pocket, however, slightly lower than in the validated binding mode of 5-HT (purple) [5].