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

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

doi:10.1074/jbc.m112.371765

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.

Highlights

The serotonin transporter contains an allosteric binding site with unknown location
The iterative approach is briefly illustrated for the serotonin transporter (SERT) configuration with (S)-CIT in the substrate binding site (S1 site) and CMI in the S2 site: (i) an (S)-CIT molecule was first docked into the S1 site of the SERT model (36); (ii) the top ranked ligand-transporter complex was reimmersed in an all-atom representation of a bilayerwater environment and was subjected to 48-ns molecular dynamics (MD) simulations; (iii) a CMI molecule was subsequently docked in the S2 site, based on an equilibrated MD snapshot from stage (ii); and (iv) an MD simulation in explicit solvent was carried out following the protocol described in (ii)
During the equilibration of our SERT model with (S)-CIT bound in both sites, when the poses of the ligands and surrounding SERT regions were stabilized, we observed the coordination of S2:(S)-CIT to include residues Leu99, Trp103, and Arg104 in TM1; Ile179 in TM3; Ala486, Val489, and Lys490 in transmembrane segment 10 (TM10); Val236 and Leu237 in extracellular loop 2 (ECL2); and Gly402 in ECL4 (Fig. 1, A and B)

Summary

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. SERT is the main pharmacological target in the treatments of major depression and anxiety disorders Both classical tricyclic antidepressants (TCAs), including, for example, clomipramine (CMI) and imipramine, and the selective serotonin reuptake inhibitors (SSRIs), including, for example, (S)-citalopram ((S)-CIT; Lexapro), sertraline (Zoloft), and fluoxetine (Prozac), exert their actions as potent inhibitors of SERT (3). It has been a sustained goal in the mechanistic study of these compounds to gain insight into the structural basis underlying their action at SERT. These structures revealed a binding site for TCAs and SSRIs in the LeuT located in an extracellular vestibule (termed the S2 site) that is ϳ13 Å

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION