Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

Per Plenge, Lei Shi, Thijs Beuming, Jerez Te, Amy Hauck Newman, Harel Weinstein, Ulrik Gether, Claus J Loland

Research output: Contribution to journalJournal articleResearchpeer-review

83 Citations (Scopus)

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.
Original languageEnglish
JournalThe Journal of Biological Chemistry
Volume287
Issue number47
Pages (from-to)39316-26
Number of pages11
ISSN0021-9258
DOIs
Publication statusPublished - 16 Nov 2012

Keywords

  • Allosteric Site
  • Antidepressive Agents, Second-Generation
  • Citalopram
  • Humans
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Protein Structure, Tertiary
  • Serotonin Plasma Membrane Transport Proteins
  • Zinc

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