Satinder K. Singh

Crystal structure of a bacterial homologue of Na(+)/Cl(-)-dependent neurotransmitter transporters.

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1–5 related to 6–10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.

Subunit arrangement and function in NMDA receptors

Excitatory neurotransmission mediated by NMDA (N-methyl-d-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1–NR2A heterodimer with glutamate and glycine. The NR2A–glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1–NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1–NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter’s syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 Å above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.

A competitive inhibitor traps LeuT in an open-to-out conformation

Secondary transporters are workhorses of cellular membranes, catalyzing the movement of small molecules and ions across the bilayer and coupling substrate passage to ion gradients. However, the conformational changes that accompany substrate transport, the mechanism by which a substrate moves through the transporter, and principles of competitive inhibition remain unclear. We used crystallographic and functional studies on the leucine transporter (LeuT), a model for neurotransmitter sodium symporters, to show that various amino acid substrates induce the same occluded conformational state and that a competitive inhibitor, tryptophan (Trp), traps LeuT in an open-to-out conformation. In the Trp complex, the extracellular gate residues arginine 30 and aspartic acid 404 define a second weak binding site for substrates or inhibitors as they permeate from the extracellular solution to the primary substrate site, which demonstrates how residues that participate in gating also mediate permeation.

Crystal structure and association behaviour of the GluR2 amino‐terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand‐gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular‐domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino‐terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily‐specific receptor assembly is not known. Here we show that AMPA receptor GluR1‐ and GluR2‐ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2‐ATD, propose mechanisms by which the ATD guides subfamily‐specific receptor assembly.

LeuT: a prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure.

Ion-coupled secondary transport is utilized by a broad range of integral membrane proteins to catalyze the energetically unfavorable movement of solute molecules across a lipid bilayer. Members of the solute carrier 6 (SLC6) family, present in both prokaryotes and eukaryotes, are sodium-coupled symporters that play crucial roles in processes as diverse as nutrient uptake and neurotransmitter clearance. The crystal structure of LeuT, a bacterial member of this family, provided the first atomic-level glimpse into overall architecture, pinpointed the substrate and sodium binding sites and implicated candidate helices and residues in the “gating” conformational changes that accompany ion binding and release. The structure is consistent with a wealth of elegant biochemical data on the eukaryotic counterparts and has for the first time permitted the construction of accurate homology models that can be directly tested experimentally. Sequence identity is especially high near the substrate and sodium binding sites and, thus, molecular insights within these regions have been substantial. However, there are several topics relevant to transport mechanism, inhibition, and regulation that structure/functions studies of LeuT cannot adequately address, suggesting the need for a eukaryotic transporter crystal structure.

Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.

Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay.

The scintillation proximity assay is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously expressed proteins, aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes, and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

Significance Most studies of neurotransmitter:sodium symporter (NSS) function and dynamics have been carried out in detergent even though the activity of these integral membrane proteins is heavily modulated by surrounding lipids. Here, we reconstituted the prokaryotic homolog LeuT into nanodiscs and subjected the preparation to hydrogen–deuterium exchange mass spectrometry to reveal a global view of the hallmarks of the transporter in two disparate conformations. The data were interpreted with the aid of molecular dynamics simulations, allowing unprecedented atomic-level insights into the dynamics of an unmodified, unlabeled NSS in a native-like lipid bilayer environment. Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

Mechanism of Paroxetine (Paxil) Inhibition of the Serotonin Transporter.

The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, we constructed SERT homology models based on the Drosophila melanogaster dopamine transporter and docked paroxetine to these models. We tested the predicted binding configurations with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. Our data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT’s substrate binding site directly depends on this pocket’s charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity.