Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na⁺/Cl⁻-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na⁺. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na⁺ over K⁺ or Li⁺, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na⁺ over K⁺ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na⁺ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.     

Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1

The crystallizations of the prokaryotic LeuT and of the eukaryotic DAT and SERT transporters represent important steps forward in the comprehension of the molecular physiology of Neurotransmitter: Sodium Symporters, although the molecular determinants of the coupling mechanism and of ion selectivity still remain to be fully elucidated. The insect NSS homologue KAAT1 exhibits unusual physiological features, such as the ability to use K⁺ as the driver ion, weak chloride dependence, and the ability of the driver ion to influence the substrate selectivity; these characteristics can help to define the molecular determinants of NSS function. Two non-conserved residues are present in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Na1 site. Thr 67 appears also to be significant since it is not conserved among NSS members, is present as threonine only in KAAT1 and in the paralogue CAATCH1 and, according to LeuT structure, is close to the amino acid binding site. Mutants of these residues were functionally characterized in Xenopus oocytes. The T67Y mutant exhibited uptake activity comparable to that of the wild type, but fully chloride-independent and with enhanced stereoselectivity. Interestingly, although dependent on the presence of sodium, the mutant showed reduced transport-associated currents, indicating uncoupling of the driver ion and amino acid fluxes. Thr 67 therefore appears to be a key component in the coupling mechanism, participating in a network that influences the cotransport of Na⁺ and the amino acid.     

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.     

Substrate binding and formation of an occluded state in the leucine transporter

Translocation through the extracellular vestibule and binding of leucine in the leucine transporter (LeuT) have been studied with molecular dynamics simulations. More than 0.1 μs of all-atom molecular dynamics simulations have been performed on different combinations of LeuT, bound substrate, and bound structural Na⁺ ions to describe molecular events involved in substrate binding and in the formation of the occluded state and to investigate the dynamics of this state. Three structural features are found to be directly involved in the initial steps of leucine transport: a Na⁺ ion directly coordinated to leucine (Na-1), two aromatic residues closing the binding site toward the extracellular vestibule (Tyr-108 and Phe-253), and a salt bridge in the extracellular vestibule (Arg-30 and Asp-404). These features account for observed differences between simulations of LeuT with and without bound substrate and for a possible pathway for leucine binding and thereby formation of the occluded LeuT binding site.     

Properties of an Inward-Facing State of LeuT: Conformational Stability and?Substrate Release

The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na⁺ ions and substrate bound suggest that one of the Na⁺ ion binding sites is fully disrupted. Release of alanine and the second Na⁺ ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.     

The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter

Background

The dopamine transporter (DAT), a member of the neurotransmitter:Na⁺ symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end.

Methodology/Principal Findings

Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates.

Conclusions/Significance

Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.     

Insights on Na+ binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+‐substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild‐type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na⁺ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di‐Na⁺ binding state is likely more efficient for conformational changes of NorM_VC toward the inward‐facing conformation than single‐Na⁺ binding state. The observation of two Na⁺ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na⁺ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.     

Ion-Releasing State of a Secondary Membrane Transporter

The crystal structure of Na⁺-coupled galactose symporter (vSGLT) reports the transporter in its substrate-bound state, with a Na⁺ ion modeled in a binding site corresponding to that of a homologous protein, leucine transporter (LeuT). In repeated molecular dynamics simulations, however, we find the Na⁺ ion instable, invariably and spontaneously diffusing out of the transporter through a pathway lined by D189, which appears to facilitate the diffusion of the ion toward the cytoplasm. Further analysis of the trajectories and close structural examination, in particular, comparison of the Na⁺-binding sites of vSGLT and LeuT, strongly indicates that the crystal structure of vSGLT actually represents an ion-releasing state of the transporter. The observed dynamics of the Na⁺ ion, in contrast to the substrate, also suggests that the cytoplasmic release of the Na⁺ ion precedes that of the substrate, thus shedding light on a key step in the transport cycle of this secondary transporter.     

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter

Glutamate transporters (GluTs) are the primary regulators of extracellular concentration of the neurotransmitter glutamate in the central nervous system. In this study, we have investigated the dynamics and coupling of the substrate and Na⁺ binding sites, and the mechanism of cotransport of Na⁺ ions, using molecular dynamics simulations of a membrane-embedded model of GluT in its apo (empty form) and various Na⁺- and/or substrate-bound states. The results shed light on the mechanism of the extracellular gate and on the sequence of binding of the substrate and Na⁺ ions to GluT during the transport cycle. The results suggest that the helical hairpin HP2 plays the key role of the extracellular gate for the substrate binding site, and that the opening and closure of the gate is controlled by substrate binding. GluT adopts an open conformation in the absence of the substrate exposing the binding sites of the substrate and Na⁺ ions to the extracellular solution. Based on the calculated trajectories, we propose that Na1 is the first element to bind GluT, as it is found to be important for the completion of the substrate binding site. The subsequent binding of the substrate, in turn, is shown to result in an almost complete closure of the extracellular gate and the formation of the Na2 binding site. Finally, binding of Na2 locks the extracellular gate and completes the formation of the occluded state of GluT.     

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na⁺-dependent reuptake of released neurotransmitters. Previous studies suggested that Na⁺-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na⁺ binding and transport (i.e. replacing Na⁺ with Li⁺ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na⁺ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na⁺ dependence. Thus, the detailed AIN generated from our method is shown to connect Na⁺ binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na⁺ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.     

Microseconds Simulations Reveal a New Sodium-binding Site and the Mechanism of Sodium-coupled Substrate Uptake by LeuT

The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na⁺- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na⁺ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na⁺ to Na1″, translocation to Na1, a second Na⁺ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe²⁵³ aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.     

Cryo‐EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity

The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na⁺‐dependent Cl⁻‐ and K⁺‐ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo‐electron microscopy structure of human NKCC1 in the substrate‐loaded (Na⁺, K⁺, and 2 Cl⁻) and occluded, inward‐facing state that has also been observed for the SLC6‐type transporters MhsT and LeuT. Cl⁻ binding at the Cl1 site together with the nearby K⁺ ion provides a crucial bridge between the LeuT‐fold scaffold and bundle domains. Cl⁻‐ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl⁻‐sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na⁺ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.     

The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters

The sodium solute symporters (SSS) and neurotransmitter sodium symporters (NSS) are two families of secondary transporters that are not related in amino acid sequence. Nonetheless, recent crystal structures showed that the Na⁺/galactose (SSS) and Na⁺/leucine (NSS) transporters have similar core structures. The structural relatedness highlights the need for classification methods for membrane protein structures based on other criteria than amino acid similarity. Here, we demonstrate that a method based on hydropathy profile alignments convincingly identifies structural similarity between the NSS and SSS families. Most importantly, the method shows that one of the largest transporter families for which a crystal structure is elusive (the amino acid/polyamine/organocation or APC superfamily), also shares the similar core structure observed for the Na⁺/galactose and Na⁺/leucine transporters. The APC superfamily contains the major amino acid transporter families that are found throughout life. Insight into their structure will significantly facilitate the studies of this important group of transporters.     

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure–function relationships of NSS family proteins. With hydrogen–deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (k_cat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.     

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na⁺/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.     

Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na⁺-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na⁺-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.     

Bioenergetics of Neurotransmitter Transport

Neurotransmitter transporters are essential components in the recycling of neurotransmitters released during neuronal activity. These transporters are the targets for important drugs affecting mood and behavior. They fall into at least four gene families, two encoding proteins in the plasma membrane and two in the synaptic vesicle membrane, although the known vesicular transporters have not all been cloned. Each of these transporters works by coupling the downhill movement of small ions such as Na⁺, Cl^–, K⁺, and H⁺ to the uphill transport of neurotransmitter. Plasma membrane transporters move the transmitter into the cytoplasm by cotransport with Na⁺. Many transporters also couple Cl^– cotransport to transmitter influx and these all belong to the NaCl-coupled family, although within the family the coupling stoichiometry can vary. Transporters for glutamate couple influx of this excitatory amino acid to Na⁺ and H⁺ influx and K⁺ efflux. Transporters in synaptic vesicles couple H⁺ efflux to neurotransmitter transport from the cytoplasm to the vesicle lumen.     

To be, or not to be two sites: that is the question about LeuT substrate binding

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.