RESUMO
The determination of the crystal structures of small-molecule transporters has shed light on the conformational changes that take place during structural isomerization from outward- to inward-facing states. Rather than using a simple rocking movement of two bundles around a central substrate-binding site, it has become clear that even the most simplistic transporters utilize rearrangements of nonrigid bodies. In the most dramatic cases, one bundle is fixed while the other, structurally divergent, bundle carries the substrate some 18 Å across the membrane, which in this review is termed an elevator alternating-access mechanism. Here, we compare and contrast rocker-switch, rocking-bundle, and elevator alternating-access mechanisms to highlight shared features and novel refinements to the basic alternating-access model.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Simulação de Dinâmica Molecular , Transporte Biológico , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.
Assuntos
Transporte Biológico Ativo , Íons , Bicamadas Lipídicas , Lipídeos , Proteínas de Membrana Transportadoras , Transporte de Íons , Íons/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Carreadoras de Solutos/metabolismoRESUMO
Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high-energy transition state of GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Transporte Biológico/fisiologia , Sistema X-AG de Transporte de Aminoácidos/genética , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Transporte Biológico/genética , Biologia Computacional/métodos , Mutação com Ganho de Função/genética , Modelos Moleculares , Pyrococcus horikoshii/genética , Pyrococcus horikoshii/metabolismo , Especificidade por Substrato/genéticaRESUMO
Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Medição da Troca de Deutério , Sequência de Aminoácidos , Sistema X-AG de Transporte de Aminoácidos/genética , Proteínas Arqueais/genética , Transporte Biológico , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Massas , Mutação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.
Assuntos
Flúor , Imageamento por Ressonância Magnética , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Espectroscopia de Ressonância Magnética , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Flúor , Histidina/química , Modelos Moleculares , Mutação , Níquel/química , Conformação Proteica , Domínios Proteicos , Pyrococcus horikoshii/químicaRESUMO
Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/química , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Ácido Aspártico/metabolismo , Pyrococcus horikoshii/química , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Acídicos/genética , Transporte Biológico , Cristalografia por Raios X , Detergentes , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Movimento , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteolipídeos/metabolismo , Sódio/metabolismo , Solventes , TermodinâmicaRESUMO
Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present high-speed atomic force microscopy (HS-AFM) observations of membrane-reconstituted GltPh at work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Microscopia de Força Atômica/métodos , Pyrococcus horikoshii/metabolismo , Sistema X-AG de Transporte de Aminoácidos/química , Proteínas Arqueais/química , Ácido Aspártico , Transporte Biológico , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sódio/metabolismoRESUMO
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Saccharomyces cerevisiae. Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of â¼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.
Assuntos
Antiporters/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Esteróis/química , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico Ativo/fisiologia , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismoRESUMO
Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons. Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane. This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods. We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Modelos Moleculares , Pyrococcus horikoshii/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Ácido Aspártico/química , Transporte Biológico , Transferência Ressonante de Energia de Fluorescência , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Pyrococcus horikoshii/química , Pyrococcus horikoshii/genética , Sódio/químicaRESUMO
Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding--enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context, allow discrimination between different binding mechanisms and guide drug design. In this review, we introduce advantages and limitations of ITC as a methodology to study molecular interactions of membrane proteins. We further describe case studies where ITC was used to analyze thermodynamic linkage between ions and substrates in ion-coupled transporters. Similar type of linkage analysis will likely be applicable to a wide range of transporters, channels, and receptors.
Assuntos
Calorimetria/métodos , Proteínas de Membrana/química , Sítios de Ligação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , TermodinâmicaRESUMO
Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, Glt(Ph), which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt(Ph) in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt(Ph) and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Pyrococcus horikoshii/química , Sistema X-AG de Transporte de Aminoácidos/genética , Sítios de Ligação , Transporte Biológico , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Modelos Moleculares , Movimento , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína , Sódio/metabolismo , Relação Estrutura-AtividadeRESUMO
The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology.
Assuntos
Proteínas Arqueais , Microscopia de Força Atômica , Pyrococcus horikoshii , Imagem Individual de Molécula , Pyrococcus horikoshii/metabolismo , Pyrococcus horikoshii/química , Cinética , Microscopia de Força Atômica/métodos , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Conformação Proteica , Modelos Moleculares , Cadeias de MarkovRESUMO
Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters' energy landscapes might be a widespread route of their functional diversification.
RESUMO
CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.
Assuntos
Proteínas de Escherichia coli , Prótons , Humanos , Microscopia Crioeletrônica , Íons/metabolismo , Canais de Cloreto/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Antiporters/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Translocator proteins (18 kDa) (TSPOs) are conserved integral membrane proteins. In both eukaryotes and prokaryotes, TSPOs interact with porphyrins, precursors of heme, and photosynthetic pigments. Here we demonstrate that bacterial TSPOs catalyze rapid porphyrin degradation in a light- and oxygen-dependent manner. The reaction is inhibited by a synthetic TSPO ligand PK11195 and by mutations of conserved residues, which affect either porphyrin binding or catalytic activity. We hypothesize that TSPOs are ancient enzymes mediating porphyrin catabolism with the consumption of reactive oxygen species.
Assuntos
Proteínas de Membrana Transportadoras/química , Catálise , Cromatografia Líquida de Alta Pressão , LigantesRESUMO
Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Pyrococcus horikoshii/química , Sódio/metabolismo , Sódio/farmacologia , Motivos de Aminoácidos , Sistemas de Transporte de Aminoácidos/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Ácido Aspártico/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Mutação/genética , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
Assuntos
Sistema X-AG de Transporte de Aminoácidos , Ácido Glutâmico , Humanos , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Transporte de Íons , Íons/metabolismo , Ácido Glutâmico/metabolismo , Sódio/metabolismo , Potássio/metabolismoRESUMO
Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.
Assuntos
Sistema X-AG de Transporte de Aminoácidos , Archaea , Sistema X-AG de Transporte de Aminoácidos/química , Archaea/metabolismo , Sítios de Ligação , Ácido Glutâmico/metabolismo , Cinética , Especificidade por SubstratoRESUMO
Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.