Steric Hindrance Mutagenesis in the Conserved Extracellular Vestibule Impedes Allosteric Binding of Antidepressants to the Serotonin Transporter

Abstract

Background:The serotonin transporter contains an allosteric binding site with unknown location. Results: We use molecular modeling, mutagenesis, and zinc site engineering to locate and inhibit the allosteric binding site. Conclusion: Data are consistent with the allosteric binding site being located in the extracellular vestibule. Significance: This opens the search for a new generation of antidepressant drugs directed toward the proposed allosteric binding site.

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

“…Overlap of high affinity inhibitor binding with the S1 site is supported by biochemical studies (38,44,47,55) and by recent structures of Drosophila (d) DAT and a LeuT/SERT hybrid co-crystallized with antidepressants (40,56). However, some DA transport inhibitors lack the charged nitrogen necessary to form the salt bridge with Asp-79 (57)(58)(59), suggesting that inhibitor binding can occur without this interaction and thus may not be limited to the S1 pocket (60). This possibility is supported by crystal structures of LeuT complexed at relatively low affinity with several selective serotonin uptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) in regions that overlap with S2 (16 -18, 56).…”

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

“…Recently, this notion was further supported by structures of the dopamine transporter (DAT) from Drosophila melanogaster and a LeuT/SERT hybrid protein co-crystallized with antidepressants (26,27). The role of the S2 binding site in substrate translocation is still a matter of debate, but it has recently been suggested that this region harbors a low-affinity allosteric binding site for antidepressants in SERT (28).…”

Structural basis for recognition of diverse antidepressants by the human serotonin transporter

“…Monoamine transporters have twelve transmembrane helices (TM) with TMs 1-5 and TMs 6-10 forming an invertedtopological repeat within which is a central binding site for substrate and ions approximately halfway across the membrane [7][8][9] . An extracellular vestibule in the outward-facing conformation 7,9,10 forms a secondary, allosteric site which, when occupied by ligands, can result in modulation of transporter activity by altering the kinetics of ligand dissociation from the central site [11][12][13] . Addictive substances such as cocaine and amphetamine bind to monoamine transporters and can either inhibit NT transport or promote NT efflux, respectively 14,15 .…”

“…It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/204859 doi: bioRxiv preprint first posted online Oct. 17, 2017; page 3 of 15 in modulation of transporter activity by altering the kinetics of ligand dissociation from the central site [11][12][13] . Addictive substances such as cocaine and amphetamine bind to monoamine transporters and can either inhibit NT transport or promote NT efflux, respectively 14,15 .…”