title: Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.
abstract: The serotonin transporter (SERT) controls synaptic serotonin levels and is the primary target for antidepressants, including selective serotonin reuptake inhibitors (e.g. (S)-citalopram) and tricyclic antidepressants (e.g. clomipramine). In addition to a high affinity binding site, SERT possesses a low affinity allosteric site for antidepressants. Binding to the allosteric site impedes dissociation of antidepressants from the high affinity site, which may enhance antidepressant efficacy. Here we employ an induced fit docking/molecular dynamics protocol to identify the residues that may be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory effects of Zn(2+) binding in an engineered site and the covalent attachment of benzocaine-methanethiosulfonate to a cysteine introduced in the extracellular vestibule. The data provide a mechanistic explanation for the allosteric action of antidepressants at SERT and suggest that the role of the vestibule is evolutionarily conserved among neurotransmitter:sodium symporter proteins as a binding pocket for small molecule ligands.
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Per
authors.initials: P
authors.lastname: Plenge
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Thijs
authors.initials: T
authors.lastname: Beuming
authors.forename: Jerez
authors.initials: J
authors.lastname: Te
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
authors.forename: Claus J
authors.initials: CJ
authors.lastname: Loland
mesh_terms: D000495:Allosteric Site; D018687:Antidepressive Agents, Second-Generation / Q000737:chemistry*; D015283:Citalopram / Q000737:chemistry*; D006801:Humans; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D018687:Antidepressive Agents, Second-Generation; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D015283:Citalopram; D015032:Zinc