SLC6 Research Articles

Unfolding the Structure of LeuT Employing Luminescence Resonance Energy Transfer

Background: Neurotransmitter sodium symporters (NSS) are located in the brain and retrieve neurotransmitters from the synaptic cleft to end synaptic transmission. Solute carier class six proteins (SCLC6) are of great pharmacological importance in terms of their localization and function. The crystal structures obtained from a bacterial homolog, the leucine transporter LeuTAa, in open to outward, occluded and open to inward conformations are present in frozen state with high resolution. Due to its close kinship with SLC6 proteins, LeuTAa serves as a paradigm for these transporters.Methods: In order to address the dynamicity of the substrate transport cycle in LeuTAa, we use the Lanthanide based resonance energy transfer (LRET) technique. This method is a spin-off of the fluorescence resonance energy transfer method according to Forster employing the introduction of the genetically encoded lanthanide binding tags (LBT) as donor elements. Exogenous cysteine residues labelled with cysteine specific fluorophores are used as acceptor elements. This technique is an alternative to address the movement of helices, with great resolution and has been employed successfully to examine potassium channels.Results: We screened for the functional LBT_mutants using the scintillation proximity assay. The LeuT_A335-LBT-G336 mutant displayed function in terms of its binding activity. Within this background, we generated cysteine mutants. To date, we have successfully measured the intramolecular distances in different LBT_LeuT_Cys mutants. Furthermore, we observed intramolecular distance changes from these purified proteins in detergent micelles.Conclusion: Our LRET measurements will help us to understand the transport cycle and help to complete the missing steps in substrate transport cycle of LeuTAa. Currently, we focus on the reconstitution of purified LeuTAa into liposomes and have our LRET measurements in a reconstituted system that allows to use more physiological ionic gradients.

Assessing the Transport Mechanism of Neurotransmitter Sodium Symporter Proteins with Molecular Dynamics

Neurotransmitter transporters, such as monoamine transporters (MATs), are membrane-bound proteins on the presynaptic terminal responsible for the reuptake of neurotransmitters from the synaptic cleft. Function of the MATs is associated with mood, emotion, and movement, among others. Dopamine transporter (DAT), a member of the MAT family, is responsible for the reuptake of dopamine (DA), a neurotransmitter responsible for pleasurable feelings. Work by Shan et. al. suggests that two DA molecules must bind before transport through DAT can occur, while other research disagrees. For this reason, the transport mechanism remains ambiguous. Using molecular dynamics simulations and a dual bilayer system with two DAT proteins, our study mimics physiological conditions at a synapse. One protein began in its occluded state, with DA and sodium bound in the S1 binding pocket. In this poster, we report the results from our simulation in which a single DA dissociated from the protein into the intracellular matrix without the need for a second bound DA. The sodium ions have yet to leave the binding pocket.

A Review of Monoamine Transporter-Ligand Interactions

Transporters of the monoamines serotonin, dopamine, and norepinephrine are plasma membrane proteins belonging to the neurotransmitter sodium symporter family (NSS). Monoamine transporters (MATs) by facilitating reuptake of neurotransmitters from the synapse into the presynaptic nerve terminal, regulate neurotransmitter chemical signaling and maintain homeostasis. MATs are targets for several psychostimulants such as cocaine and amphetamine; and also for drugs treating several psychiatric disorders such as depression, attention deficit hyperactivity disorder, Parkinson's disease, and schizophrenia. Since, currently available treatment has several limitations and side effects, novel treatment is highly desired. Efforts to develop better treatment have been hampered by the lack of crystal structures for MATs. However, leucine transporter (LeuTAa), a bacterial protein from Aquifex aeolicus, belonging to the same NSS family as MATs has recently been crystallized. LeuTAa is used as a template to develop homology models of MATs, which facilitates understanding of the structure, function and pharmacology of MATs. Experimental methods for drug discovery demand a significant amount of time, effort and money. Efficient utilization of computational techniques hastens the process of drug discovery and also significantly reduces the cost. Assessing the binding affinity of drugs to the receptors is a key aspect of drug design. Free energy calculations compliment the experiment by quantitatively assessing the affinity of ligands to receptors. These methods are highly beneficial in the lead identification and optimization stages of rational drug design. We review the currently available free energy methods to treat protein-ligand interactions along with several free energy studies performed on MATs.

The Membrane Protein LeuT in Micellar Systems: Aggregation Dynamics and Detergent Binding to the S2 Site

Structural and functional properties of integral membrane proteins are often studied in detergent micellar environments (proteomicelles), but how such proteomicelles form and organize is not well understood. This makes it difficult to evaluate the relationship between the properties of the proteins measured in such a detergent-solubilized form and under native conditions. To obtain mechanistic information about this relationship for the leucine transporter (LeuT), a prokaryotic homologue of the mammalian neurotransmitter/sodium symporters (NSSs), we studied the properties of proteomicelles formed by n-dodecyl-β,d-maltopyranoside (DDM) detergent. Extensive atomistic molecular dynamics simulations of different protein/detergent/water number ratios revealed the formation of a proteomicelle characterized by a constant-sized shell of detergents surrounding LeuT protecting its transmembrane segments from unfavorable hydrophobic/hydrophilic exposure. Regardless of the DDM content in the simulated system, this shell consisted of a constant number of DDM molecules (∼120 measured at a 4 Å cutoff distance from LeuT). In contrast, the overall number of DDMs in the proteomicelle (aggregation number) was found to depend on the detergent concentration, reaching a saturation value of 226±17 DDMs in the highest concentration regime simulated. Remarkably, we found that at high detergent-to-protein ratios we observed two independent ways of DDM penetration into LeuT, both leading to a positioning of the DDM molecule in the second substrate (S2) binding site of LeuT. Consonant with several recent experimental studies demonstrating changes in functional properties of membrane proteins due to detergent, our findings highlight how the environment in which the membrane proteins are examined may affect the outcome and interpretation of their mechanistic features.

X-ray structure of dopamine transporter elucidates antidepressant mechanism

Antidepressants targeting Na(+)/Cl(-)-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses.

Coupled Global and Local Changes Direct Substrate Translocation by Neurotransmitter-Sodium Symporter Ortholog LeuT

Significant advances have been made in recent years in characterizing neurotransmitter:sodium symporter (NSS) family structure and function. Yet, many time-resolved events and intermediates that control the various stages of transport cycle remain to be elucidated. Whether NSSs harbor one or two sites for binding their substrates (neurotransmitters or amino acids), and what the role of the secondary site S2 is, if any, are still unresolved. Using molecular modeling and simulations for LeuT, a bacterial NSS, we present a comprehensive account of substrate-binding and -stabilization events, and subsequently triggered interactions leading to substrate (alanine) release. LeuT instantaneous conformation as it reconfigures from substrate-receiving (outward-facing) to -releasing (inward-facing) state appears to be a determinant of its affinity to bind substrate at site S2. In the outward-facing state, S1 robustly binds alanine and regulates subsequent redistribution of interactions to trigger extracellular gate closure; whereas S2 is only a transient binding site. The substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. LeuT harbors the two substrate-binding sites, and small displacements of second substrate near S2 are observed to induce concerted small translocations in the substrate bound to primary site S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm.

Chloride binding site of neurotransmitter sodium symporters

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

Pairwise structural comparison of tiagabine analogs gives new insights into their protein binding modes

Tiagabine (Gabitril®) is a selective inhibitor of the human gamma-aminobutyric acid (GABA) transporter 1 (hGAT-1), a transport protein belonging to the family of neurotransmitter-sodium-symporters (NSS). It is a marketed drug, used for treatment of epilepsy. However, the molecular basis of protein-ligand interaction remains obscure due to the lack of a 3D structure of the target protein. In order to identify activity-determining structural features of a series of tiagabine analogs taken from literature [1-3], we chose an approach combining traditional methods of molecular modeling with exhaustive sampling of docking poses, and a pairwise comparison of structural features and their respective bioactivity values. We determined a common binding mode of tiagabine analogs, which is in nice agreement with literature [4]. Further, we were able to trace back considerable differences in inhibitory activities to distinct molecular attributes of the analogs. Our study revealed the molecular explanation for the importance of a polar linker region and thus paves the way for subsequent screening efforts in the search for novel GAT-1 inhibitors.

The cost of living in the membrane: A case study of hydrophobic mismatch for the multi-segment protein LeuT

Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid–protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane–protein interface from MD simulations. LeuT has been serving as a model for structure–function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar–hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar–hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic–hydrophilic interactions hinder the motion of TM1a in the functionally relevant conformational transition to the inward-open state. Various extents of such unfavorable interactions, involving exposure to the lipid environment of adjacent hydrophobic and polar residues, are common in multi-segment transmembrane proteins, and must be considered to affect functionally relevant conformational transitions.

Fluorometric Characterization of Substrate Binding in a Bacterial NSS Homologue

The prokaryotic amino acid transporter LeuT belongs to the neurotransmitter:sodium symporter (NSS) family that also includes mammalian transporters of neurotransmitters such as dopamine, serotonin and nor-epinephrine. NSS are key terminators of synaptic transmission in CNS, and are the targets of various therapeutics as well as drugs of abuse. Here we seek to further elucidate the molecular details of the sodium dependent translocation cycle by which substrate is transported across the cell membrane. To this end, we have introduced the environmentally sensitive fluorophore tetramethylrhodamine to the cytosolic end of TM5 in LeuT a region shown in mammalian NSS to undergo change in solvent accessibility during substrate translocation. Fluorescent labelling of the single cystein mutant yields a functional transporter. With fluorescence spectroscopy, we then demonstrate that binding of a transported substrate induces a sodium dependent increase in accessibility of the fluorophore to the hydrophilic quencher iodide (KI). Tryptophan, a competitive inhibitor of WT LeuT, only induces an increase in fluorophore accessibility when the change of substrate specificity mutation I359Q is introduced. Finally, addition of the tricyclic antidepressant clomipramine (CMI) only increases the apparent accessibility in the presence of a transported substrate. Collectively, our findings represent a direct measure of substrate bound occluded state formation, a conformational step preceding transporter isomerization to the inward facing conformation.

Allostery in the Ion-Controlled Conformational Dynamics of LeuT

Neurotransmitter: sodium symporter (NSS) proteins are secondary Na+-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs - Leucine Transporter (LeuT) - and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na+ binding site (Na1 site). In the course of MD simulations started from the occluded state with bound Na+, but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu290. From the calculations we identified as well a quasi-stable Na+ binding site (Na1' site), in which Na+ is in direct ionic interaction with Glu290. The occupancy of the Na1' site is associated with the modulation of conformational transition towards outward opening in response to different perturbations. Examples include Li+ replacing Na+ in the Na sites, and the effects of the Y268A mutation that was known to shift the inward/outward conformational equilibrium. As the computational study tracks the remarkable changes in the extent of outward opening, we were able to identify the allosteric elements that are critical in mediating the impact of the perturbations on the conformational transition. Together, these findings shed new light on the Na+-driven transport cycle and on the dynamic details of the nature and role of allostery in the structural rearrangements associated with outward- and inward-open transitions.

Decrypting structural and functional changes in LeuTAaat atomic level employing LRET

Background Neurotransmitter:sodium symporters (NSS) are integral membrane proteins that mediate the reuptake of monoamine neurotransmitters previously released into the synaptic cleft. They are of pharmacological significance because they are the target of many clinically important drugs. LeuTAa, a leucine/alanine transporter is a bacterial homolog to NSS. Crystal structures of LeuTAa with open to outward, occluded and inward-facing states have already been resolved at high resolution. Hence, LeuTAa serves as a good paradigm for exploring the structurefunction relationship of NSS proteins.

Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na+-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs—Leucine Transporter (LeuT)—and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na+ binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na+, but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na+ affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na+ over K+ in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na+-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

Insights into binding events of GABA- and Tiagabine- analogues in the Gamma-Aminobutyric Acid Transporter 1 by means of Molecular Modelling

The human transporters for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) hGAT-1, 2, and 3, and hBGT-1 belong to the neurotransmittersodium symporter (NSS) family of membrane transport proteins. hGAT-1 has been a target for the design of antiepileptic therapeutics [1], with tiagabine (Gabitril ® ) being the only GAT inhibitor on the market. The lack of specific inhibitors for the other hGAT subtypes, results from a still missing detailed understanding of the molecular basis of drug-transporter interactions of the respective subtypes. We aim at elucidating plausible binding modes for ligands of each subtype, respectively. In our first studies, we built up homology models for hGAT-1 in the occluded and outward-facing conformation based on the respective high resolution structures of the leucine transporter of Aquifex aeolicus (LeuT) (pdb-codes: 2A65 and 3F3A). Afterwards, the natural substrate GABA was docked into the occluded state model and tiagabine into the outward-facing model by making use of the Induced Fit Docking module of Schrodinger, LLC. Both models were further subject to extensive Molecular Dynamics (MD) simulation studies (GROMACS 4.5.3 was used). By the aid of MD simulations we could detect the existence of conserved water molecules into the GABA (occluded) and tiagabine (open-to-out) binding sites, respectively. Average structures from the equilibrated trajectories were extracted and subsequently served for further docking experiments. Docking of small ligands (GABA, Guvacine and R-/S Nipecotic Acid) into the occluded state model nicely indicated their preference to bind in an extended conformation (also demonstrated by long-term MD with GABA). MD and Docking of tiagabine and analogues into the open-to-out state model revealed a common binding mode. Additionally, our studies will be extended to the other hGAT subtypes so as to elucidate the secret of subtype selectivity of GABA Transporters.

Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

X-ray structures of LeuT in substrate-free outward-open and apo inward-open states

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.

Structure, Dynamics, and Mechanism of the Leucine Transporter Studied by Double Electron Electron Resonance Spectroscopy

The neurotransmitter/sodium symporter (NSS) family of transporters includes the human dopamine, serotonin, and norepinephrine transporters. These transporters harness sodium and chloride gradients to facilitate reuptake of neurotransmitters from the synapse thus terminating neurotransmission. Due to their critical regulatory role, NSSs are the targets of numerous psychiatric therapeutics and drugs of abuse. Progress in understanding and treating diverse conditions including depression and anxiety will require a detailed description of the structure and function of this class of proteins. Structural investigations of NSS have focused on a bacterial homolog of these transporters, LeuT. This work will describe a systematic investigation of the intracellular dynamics of LeuT as well as detail progress in defining the LeuT transport mechanism and open-inward structure. Using double electron-electron resonance (DEER) spectroscopy, a pulsed EPR technique, over 40 double mutants were analyzed for their distance distributions and relative dynamics. Mechanistic descriptions were inferred by tracking shifts in equilibria between multi-component distance distributions and relating these shifts to biochemical conditions across all mutants. Closed-inward distances were largely consistent with the LeuT crystal structures, while primary gating motions were identified at helices 1 and 6 with additional reorientations of helices 5 and 7, resulting in the open-inward structure. Distance data are being implemented into restrained computational modeling to generate preliminary models of the open-inward conformation of LeuT. These results will be compared to the current models of LeuT open-in structure and transport mechanism derived using alternative approaches.

Templates and Models of Monoamine Transporter Proteins

X-ray crystallography, structural bioinformatics and computational chemistry have become important techniques in the discovery and development of effective and safe new drugs. From a drug discovery point of view, membrane proteins are among the most interesting molecular targets, but the current knowledge about detailed 3D structures of membrane proteins is sparse. Homology modeling techniques may provide structural knowledge about membrane proteins and their interactions with drugs and other molecules. The neurotransmitter sodium symporters (NSS) are the molecular targets of many pharmacologically active substances, and we have used three different secondary transporters as templates for modeling the NSS proteins DAT, NET and SERT. The first template was based on the electron density projection map of the Escherichia coli Na+/H+ antiporter (NhaA), while later the X-ray structure of Lac Permease (symporter) was used as a template. The helical architectures of these templates have a lot in common, and models based on both could contribute with structural explanations of several experimental studies in spite of low homology with NSS proteins. In 2005 the crystal structure of a bacterial homologue of the human monoamine neurotransmitter transporter Aquifex aeolicus (LeuTAa) was reported. This structure was the first experimental structure of a NSS family member, and represented a breakthrough for homology modeling of pharmacological important NSS proteins. Since then several X-ray structures LeuTAa in complex with pharmacologically important compounds have been published. Homology models of NSS proteins, combined with site-directed mutagenesis data, have identified ligand binding sites and contributed with important knowledge for new drug development.