Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter
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Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter. / Plenge, Per; Shi, Lei; Beuming, Thijs; Te, Jerez; Newman, Amy Hauck; Weinstein, Harel; Gether, Ulrik; Loland, Claus J.
In: The Journal of Biological Chemistry, Vol. 287, No. 47, 16.11.2012, p. 39316-26.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter
AU - Plenge, Per
AU - Shi, Lei
AU - Beuming, Thijs
AU - Te, Jerez
AU - Newman, Amy Hauck
AU - Weinstein, Harel
AU - Gether, Ulrik
AU - Loland, Claus J
PY - 2012/11/16
Y1 - 2012/11/16
N2 - 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.
AB - 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.
KW - Allosteric Site
KW - Antidepressive Agents, Second-Generation
KW - Citalopram
KW - Humans
KW - Molecular Docking Simulation
KW - Molecular Dynamics Simulation
KW - Mutagenesis
KW - Protein Structure, Tertiary
KW - Serotonin Plasma Membrane Transport Proteins
KW - Zinc
U2 - 10.1074/jbc.M112.371765
DO - 10.1074/jbc.M112.371765
M3 - Journal article
C2 - 23007398
VL - 287
SP - 39316
EP - 39326
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 47
ER -
ID: 47292422