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

2017 ◽  
Author(s):  
Jonathan A. Coleman ◽  
Eric Gouaux
Keyword(s):  
Binding Site ◽  
Serotonin Transporter ◽  
Serotonin Reuptake ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Structural Basis ◽  
Local Concentration ◽  
Substrate Binding Site ◽  
X Ray ◽  
Open Conformation ◽  
Drug Selectivity

ABSTRACTSelective serotonin reuptake inhibitors are clinically prescribed antidepressants that act by increasing the local concentration of neurotransmitter at synapses and in extracellular spaces via blockade of the serotonin transporter. Here we report x-ray structures of engineered thermostable variants of the human serotonin transporter bound to the antidepressants sertraline, fluvoxamine, and paroxetine. The drugs prevent serotonin binding by occupying the central substrate binding site and stabilizing the transporter in an outward-open conformation. These structures explain how residues within the central site orchestrate binding of chemically diverse inhibitors and mediate transporter-drug selectivity.

scholarly journals Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

2020 ◽  
Author(s):  
Lan Guan ◽  
Parameswaran Hariharan
Keyword(s):  
Molecular Recognition ◽  
Binding Site ◽  
Crystal Structures ◽  
Mammalian Cells ◽  
Substrate Binding ◽  
Structural Basis ◽  
Cooperative Binding ◽  
Recognition Mechanism ◽  
Substrate Binding Site ◽  
X Ray

AbstractThe symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

scholarly journals Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Zachary Lee Johnson ◽  
Jun-Ho Lee ◽  
Kiyoun Lee ◽  
Minhee Lee ◽  
Do-Yeon Kwon ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Nucleoside Transporters ◽  
Structural Basis ◽  
Biological Processes ◽  
Functional Information ◽  
Binding Studies ◽  
X Ray ◽  
Equilibrium Binding ◽  
Drug Selectivity ◽  
Proof Of Principle

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.

scholarly journals Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

2020 ◽  
Author(s):  
Shabareesh Pidathala ◽  
Aditya Kumar Mallela ◽  
Deepthi Joseph ◽  
Aravind Penmatsa
Keyword(s):  
Chronic Pain ◽  
Binding Site ◽  
Dopamine Transporter ◽  
Binding Pocket ◽  
Norepinephrine Transporter ◽  
Structural Basis ◽  
List Type ◽  
X Ray ◽  
Neurotransmitter Transporters ◽  
Reuptake Inhibition

AbstractNorepinephrine is a biogenic amine neurotransmitter that has widespread effects on cardiovascular tone, alertness and sensation of pain. As a consequence, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ a modified Drosophila melanogaster dopamine transporter as a surrogate for the human norepinephrine transporter and determine the X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.HighlightsX-ray structures of the Drosophila dopamine transporter in substrate-free and norepinephrine bound forms.Norepinephrine and dopamine bind in distinct conformations within the binding pocket.Chronic pain inhibitors S-duloxetine, milnacipran and tramadol bind in the primary binding site and overlap with the norepinephrine-binding pose.Selective norepinephrine reuptake inhibition occurs through specific interactions at the subsite C in the primary binding pocket.

scholarly journals Use of selective serotonin reuptake inhibitors is associated with very low plasma-free serotonin concentrations in humans

2019 ◽  
Vol 57 (1) ◽  
pp. 59-63
Author(s):  
Marloes AM Peters ◽  
Martijn van Faassen ◽  
Wilhelmina HA de Jong ◽  
Grietje Bouma ◽  
Coby Meijer ◽  
...  
Keyword(s):  
Serotonin Transporter ◽  
Serotonin Reuptake ◽  
High Speed ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Plasma Concentrations ◽  
Serotonin Reuptake Inhibitors ◽  
Selective Serotonin ◽  
Blood Collection ◽  
Plasma Tryptophan ◽  
Platelet Serotonin

Background Selective serotonin reuptake inhibitors (SSRIs) block the serotonin transporter on neurons, but also on platelets, thus decreasing platelet serotonin concentrations in users of SSRIs. Data on plasma-free serotonin concentrations in SSRI users are lacking, while plasma-free serotonin is available for receptor binding and plays a role in several pathophysiological processes. We therefore measured the plasma-free and platelet serotonin concentrations in users of SSRIs and age-matched healthy controls, and we analysed plasma concentrations of the serotonin precursor tryptophan and serotonin metabolite 5-hydroxyindoleamineacetic acid (5-HIAA). Methods For this cross-sectional single-centre case control study, participants were recruited at the departments of Psychiatry and General Medicine. High-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to measure plasma-free and platelet serotonin, plasma tryptophan and 5-HIAA concentrations. Preanalytical conditions were optimized by careful blood collection, rapid sample handling, high-speed centrifugation, drug and diet restrictions and age-matched controls. Results In 64 SSRI users, median concentrations of plasma-free and platelet serotonin were 10-fold and 14-fold lower, respectively, than in 64 matched controls. Patients using higher dose SSRIs or those with higher affinity for the serotonin transporter had lower plasma-free and platelet serotonin concentrations. Compared with controls, SSRI users had similar median plasma tryptophan concentrations but slightly higher plasma 5-HIAA concentrations. Conclusion SSRI users have low platelet serotonin and low plasma-free serotonin. This could not be explained by lower concentrations of its precursor tryptophan, and only partially by increased breakdown to 5-HIAA.

scholarly journals A Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells

2021 ◽  
Vol 15 ◽  
Author(s):  
Ian D. Tomlinson ◽  
Oleg Kovtun ◽  
Ruben Torres ◽  
Laurel G. Bellocchio ◽  
Travis Josephs ◽  
...  
Keyword(s):  
Quantum Dot ◽  
Binding Site ◽  
Serotonin Transporter ◽  
Reuptake Inhibitor ◽  
Serotonin Reuptake ◽  
Membrane Dynamics ◽  
Biologically Active ◽  
Therapeutic Effects ◽  
Serotonin Reuptake Inhibitor ◽  
Hek 293

The serotonin transporter (SERT) is the primary target for selective serotonin reuptake inhibitor (SSRI) antidepressants that are thought to exert their therapeutic effects by increasing the synaptic concentration of serotonin. Consequently, probes that can be utilized to study cellular trafficking of SERT are valuable research tools. We have developed a novel ligand (IDT785) that is composed of a SERT antagonist (a tetrahydro pyridyl indole derivative) conjugated to a biotinylated poly ethylene glycol (PEG) via a phenethyl linker. This compound was determined to be biologically active and inhibited SERT-mediated reuptake of IDT307 with the half-maximal inhibitory concentration of 7.2 ± 0.3 μM. We demonstrated that IDT785 enabled quantum dot (QD) labeling of membrane SERT in transfected HEK-293 cultures that could be blocked using the high affinity serotonin reuptake inhibitor paroxetine. Molecular docking studies suggested that IDT785 might be binding to the extracellular vestibule binding site rather than the orthosteric substrate binding site, which could be attributable to the hydrophilicity of the PEG chain and the increased loss of degrees of freedom that would be required to penetrate into the orthosteric binding site. Using IDT785, we were able to study the membrane localization and membrane dynamics of YFP-SERT heterologously expressed in HEK-293 cells and demonstrated that SERT expression was enriched in the membrane edge and in thin cellular protrusions.

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