Research Article: A structural model of the human serotonin transporter in an outward-occluded state

Date Published: June 28, 2019

Publisher: Public Library of Science

Author(s): Eva Hellsberg, Gerhard F. Ecker, Anna Stary-Weinzinger, Lucy R. Forrest, Claudio M. Soares.


The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

Partial Text

The serotonin transporter hSERT belongs to the secondary active solute carrier 6 (SLC6) membrane protein family, in which it forms the subgroup of monoamine transporters (MAT) together with the dopamine and norepinephrine transporters, DAT and NET [1,2]. The SLC6 transporters are human proteins belonging to the larger family of neurotransmitter:sodium symporters (NSS; Transporter Classification Database [3] identifier 2.A.22). The solute transported by hSERT is serotonin (5-hydroxytryptamine, 5HT), an important tissue hormone in the periphery and a neurotransmitter in the central nervous system. In its neurotransmitter function, 5HT plays a crucial role in regulation of, or impact on, mood, sleep-wake cycle, appetite, pain, sexuality, and body temperature control. hSERT is the primary target for competitive inhibitors in major depression therapy, but it also interacts with inhibiting, or even transport-reverting, psychostimulants. According to the WHO, depression is globally the leading cause of disability [4]. Despite its importance, the serotonin transport mechanism is not yet fully understood. 5HT is released from vesicles in presynaptic neurons into the synaptic cleft, where it transmits its signal to the postsynaptic 5HT-receptors. After transmission, 5HT is taken back into presynaptic neurons for degradation or vesicle storage. This reuptake against its concentration gradient is facilitated by hSERT under cotransport of sodium [5,6]. Furthermore, chloride is required for transport activity [7], while potassium antiport stimulates the transport process [8]. However, the exact transport stoichiometry remains elusive and the potential binding site for potassium in the transporter is unknown; both questions need to be addressed for a complete understanding of the transport mechanism. To facilitate 5HT reuptake, the transporter needs to undergo distinct conformational changes. In principle, these changes expose the substrate binding site(s) to one side of the membrane at a time, according to the so-called alternating-access mechanism [9], support for which has been provided by structural modeling and biochemical experiments [10–12], and recently also by X-ray and cryo-EM crystallography [13–15]. Taken together, the hSERT transport cycle can be represented roughly as shown in Fig 1, albeit with the caveat that several important details remain elusive.

In this study, we have built a comparative model of hSERT in an outward-occluded state based on structural information primarily from the resolved hSERT X-ray structure itself. This was accomplished by fitting the upper bundle parts to the corresponding region in LeuTAa 3F48 before using them as a template combined with the remaining parts of the outward-open hSERT (5I71). We emphasized the model selection and refinement steps, and integrated knowledge from 5I71, 5I6X, and 3F48 to the greatest degree possible. The docked orientation of 5HT in its orthosteric binding site in both the outward-occluded model and the outward-open X-ray structure 5I71 were in excellent agreement with experimentally-validated induced-fit docking results obtained previously for a LeuTAa-based model of hSERT [45]. Consequently, we conducted microsecond-long MD simulations of the ion- and substrate-bound complex, embedded in a membrane, to predict structural features that characterize the outward-occluded state. Simulations of the 5HT-bound outward-open structure of hSERT were used as a control.