The substrate import mechanism of the human serotonin transporter

Abstract

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states have provided a glimpse of the functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to intermediate states are not fully understood. Furthermore, the molecular basis of how serotonin is recognized and transported remains unclear. In this study, we performed unbiased microsecond-long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational-driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We find that the transition from the occluded to inward-facing state is the rate-limiting step for substrate import and that the substrate decreases the free energy barriers to achieve the inward-facing state. Our study provides insights on the molecular basis of dynamics-driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Figure 1

Conformational free energy landscapes of SERT obtained from MD simulations. Relative free energies from MSM-weighted simulation data plotted against the distances between extracellular and intracellular gates for (A) Na⁺-SERT and (B) 5-HT-SERT. The OF SERT crystal structure (PDB: 5I73) was used as the starting structure for MD simulations and transitioned to OC and IF states. An HG state, in which both gates are open, was also observed. The SERT structure is represented as cartoon with TM1, 5, 6, 8, and 10 colored in teal, green, magenta, yellow, and orange respectively. (C) Cross section through SERT conformational states viewed from the membrane plane, shown as surface representations. The channel pore volume across the transporter is depicted as dark blue spheres and extracellular gates Arg104 and Glu493 are shown as teal and orange sticks, respectively. To see this figure in color, go online.

Figure 2

MD analysis of global fluctuations during conformational transitions. RMSF of Na⁺ (A and B) and 5-HT transport (C and D) for OF to OC transition and OC to IF transition mapped to the SERT structure. Tube thickness corresponds to the RMSF values of each residue. To calculate the RMSF between transitions, 20,000 structures of OC and IF states were randomly extracted from the conformational landscape and measured with respect to the cryo-EM structure of the prior conformational state. The simulations show fluctuations involving EL4, EL6, and TM5 to be coupled with substrate import. More specifically, EL4 and EL6 experience greater dynamics in the presence of the 5-HT when SERT transitions from OF to OC. Compared with Na⁺-SERT simulations, the EL dynamics are less pronounced. In OC to IF transitions of 5-HT-SERT, EL2 is stabilized while the fluctuations at cytoplasmic base of TM5 increase to form the intracellular exit pathway. Alternatively, in Na⁺-SERT, EL2 is destabilized and TM5 dynamics are reduced. To see this figure in color, go online.

Figure 3

(A and B) MD snapshots of the hydrogen bonding network involving buried glutamate residues and water molecules when SERT adopts the OF state (A) and the IF state (B). TM helices 2, 6, and 10 colored as pale blue, magenta, and orange respectively. (C) A third Na⁺ ion binding site buried beneath the orthosteric pocket as a result of Glu508 deprotonation. MD snapshot (TM2 colored pale blue; TM6, magenta; TM10, orange) superimposed with the SERT crystal structure (PDB: 5I73, gray), with electron density shown at 0.5 σ. To see this figure in color, go online.

Figure 4

The major flux pathway and MFPTs for SERT conformational transitions and 5-HT import determined from transition path theory. The transport process begins with the binding of two Na⁺ ions to the Na1 and Na2 sites in the OF state (2, 3). 5-HT diffuses to the orthosteric site (4). Next, a Cl⁻ ion, accompanied by an additional Na⁺ (not shown), enters the transporter and binds (5). The accompanying Na⁺ ion dissociates back to the extracellular space (see Fig. S33 for additional details). The binding of the substrate and ions facilitates the closure of the extracellular gate to form the OC state (6). Isomerization to the IF state is associated with the release of Na⁺ from the Na2 site and 5-HT diffuses out (6–9). Arrow thickness represents relative flux between transitions. To see this figure in color, go online.

Figure 5

MD snapshots of the simulated mechanism for SERT-catalyzed 5-HT import. (A) Overlaid MD snapshots of 5-HT translocation, from when 5-HT enters the extracellular vestibule (blue) to its cytosolic exit (red). The positions of ions in the OC state are shown as spheres. TM helices 1, 5, 6, 8, and 10 are colored in teal, green, magenta, yellow, and orange, respectively. (B) 5-HT enters the transporter by binding in the allosteric site. (C) From the allosteric site, 5-HT rotates down the transport pathway to the orthosteric site. (D) Initial Cl⁻ recognition is assisted by Asp98, Arg104, Tyr175, Phe335, and indole of 5-HT. To see this figure in color, go online.

Figure 6

MD snapshots of 5-HT in the orthosteric binding site and intracellular release. TM helices 1, 5, 6, 8, and 10 are colored in teal, green, magenta, yellow, and orange, respectively. Substrate color relates to progression of 5-HT-import as shown in Fig. 5A. (A and B) While 5-HT is in subsites C and B, the amine moiety of 5-HT at the orthosteric site interacts with Asp98, disrupting the hydrogen bonding interaction between Tyr176-Asp98. (C) The rotameric flip of the phenol ring of Tyr95 initiates the opening of the intracellular vestibule and allows for permeation of 5-HT toward the intracellular exit pathway. (D) 5-HT translocation through the exit pathway between TM1a and TM5. To see this figure in color, go online.

Figure 7

Cross-correlation analysis of Na⁺-SERT and 5-HT-SERT simulations. (A) The cross-correlation matrix depicts coupled motion between structural elements of the transporter. The upper left triangle of the matrix contains correlation values in 5-HT-SERT simulations, while the lower right triangle is from Na⁺-SERT simulations. The presence of the substrate increases the cooperative dynamics of the gating helices, specifically the motions of TM1a and TM10 (circled in red), while these motions are less pertinent in Na⁺-SERT. Additionally, greater correlated motions of TM12 with TM5 and TM7 are present with the addition of the substrate (circled in green), while motions of TM2 show higher correlations without substrate (circled in magenta). (B and C) Positive cross-correlation relationships between residues plotted as pale red lines on the SERT tertiary structure. The correlation of the gating helices in the presence of the substrate is circled in bold red in (C). To see this figure in color, go online.