Prozac and its kin — drugs called selective serotonin reuptake inhibitors (SSRIs) — were first discovered in 1972. They address one hallmark of depression: low levels of the molecule serotonin, which neurons use to signal one another. By preventing a protein called serotonin transporter (SERT) form absorbing the serotonin back into neurons that release it, the drugs boost serotonin levels in the junctions between cells. It is exciting that the structure of the serotonin transporter targeted by several widely used antidepressants — has been solved. The finding, reported on 6 April in Nature (Coleman, J. A., Green, E. M. & Gouaux, E. Nature http://dx.doi.org/10.1038/nature17629 (2016)). Also, Creative Biomart provides SERT (SLC6A4) for research application when you need. Here we’d like to share this wonderful article.

Abstract: The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.

Architecture of human SERT: The structure of human SERT bound to (S)-citalopram or paroxetine exhibits an outward-open conformation with the antidepressant drug bound to the central site, halfway across the membrane and wedged into a cavity made up of residues from TM1, TM3, TM6, TM8 and TM10 (Fig. b, c). A second (S)-citalopram molecule was found in the allosteric site, within the extracellular vestibule of the (S)-citalopram cocrystal structure, approximately 13 Å from the central site. Akin to dDAT and LeuT, SERT has 12 transmembrane-spanning helices with TM1–TM5 and TM6–TM10 related by a pseudo-two-fold axis. The ts2 and ts3 transporters superimpose well, demonstrating that the additional mutation of the ts3 construct does not substantially perturb the functionally active ts2 transporter structure. TM1 and TM6 adopt short regions of non-helical conformation as they skirt the central ligand site and contribute residues that bind inhibitors as well as coordinate Na⁺ and Cl⁻ ions. The conformations of TM1 and TM6 are incompatible with the formation of an occluded state, suggesting that the antidepressant molecules have locked the transporter in an outward-open conformation, similar to the inhibitor-bound outward-open conformations of dDAT and LeuT.

The extracellular surface of SERT is largely composed of extracellular loop (EL) 2, EL4 and EL6, with EL2 ‘combed-over’ the extracellular surface and providing 3,376 Å of solvent-accessible surface area. A conserved disulfide bridge is formed between Cys200 and Cys209 in EL2 (ref. 27). EL2 is predicted to contain two N-linked glycosylation sites, Asn208 and Asn217 (ref. 28), and electron density for a N-acetylglucosamine moiety was found linked to Asn208; weak density was also found near Asn217. Similar to dDAT, the intracellular surface of the transporter is capped by intracellular loop (IL) 1, IL5 and the carboxy-terminal helix. Unlike LeuT, yet reminiscent of dDAT, TM12 has a pronounced kink halfway across the membrane. There is a cholesterol hemisuccinate (CHS) molecule bound near TM12a.

The crystal lattice packing between two SERT molecules occurs at the kink in TM12, which also overlaps with a two-fold axis of crystallographic symmetry, thus generating an apparent SERT ‘dimer’. Experiments suggest that SERT is an oligomer in the membrane. However, in detergent SERT is a monomer and we suggest that the SERT ‘dimer’ observed in this crystal form is unlikely to exist in a membrane bilayer because the predicted membrane-spanning regions of each protomer are not aligned with one another. Because the electron density for the Fab constant domain was poor, we also solved the structure of the Fab at 1.6 Å resolution to facilitate model building and refinement. The Fab binds to a large extracellular surface consisting of EL2 and EL4 in a symmetry-related SERT, and this interface is further stabilized by interactions of EL2–EL2 and Fab–EL2 in the asymmetric unit.

The structure of SERT shows that amino acid changes due to single nucleotide polymorphisms and mutations associated with psychiatric disorders are distributed throughout the structure. Interestingly, most of the altered residues face solvent or lipid, thus rendering their effect on SERT structure and function obscure. Pro339Leu, however, is located in the non-helical region of TM6 neighbouring the ligand-binding site and, not surprisingly, this variant exhibits diminished transport activity. By contrast, other disease-associated mutations and polymorphisms, including mutations at Ile425 in TM8, Lys201Asn in EL2 (ref. 30) and Ser293Phe and Leu362Met in TM5 and TM7 enhance serotonin transport, respectively. Another class of mutations, Phe465Leu in TM9 and Leu550Val in TM11, probably destabilize the transporter or, as in the case of the Lys605Asn substitution in the C-terminal helix, render the transporter insensitive to protein kinase G regulation. With the establishment of SERT structural analysis, together with SERT expression and purification, we can now determine more precisely how these mutations alter the structure and activity of SERT.

Intracellular surface and C-terminal hinge: IL5 and the intracellular half of TM11 are highly similar to dDAT, while IL4 is partially unwound due to the insertion of Trp458. The C terminus of SERT mimics dDAT with a similar hinge and helix region. Glu615 is thought to form a salt bridge with Arg152 in IL1 (ref. 49), but no side-chain density is present, which makes assignment of C-terminal register not possible. We propose that the disorder of the C terminus is due to dynamic properties, perhaps related to its importance in trafficking.

Conclusion: The SERT–SSRI complexes capture the transporter in an inhibitor bound, outward-open conformation, illustrating how the bulky ligands lodge in the central binding site, preventing substrate binding and transporter isomerization to occluded and inward-open conformations. Extensive interactions throughout the central binding site explain, in large part, the selectivity of SSRIs. The allosteric site is poised ‘above’ the central site, within the ‘walls’ of the extracellular vestibule, directly obstructing ligand egress from the central site, thus explaining how allosteric ligands slow the off-rate of inhibitors bound to the central site. Taken together, the structures of the human serotonin transporter shed fresh insight into antidepressant recognition and the molecular basis for allosteric modulation of inhibitor binding and of transporter activity, thus providing a platform to design small molecules targeting the central and allosteric binding sites.

For more details please visit  http://dx.doi.org/10.1038/nature17629.