10803966
The structural basis of secondary active transport mechanisms.
Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.
9935446
Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier.
Abstract Two models for mediated transport are described. The “allosteric” model involves transition of a combining site between two alternate shapes. The “H bond” model involves transition of a combining site between two alternate hydrogen bonded states. In both cases, the alternate forms have different directional properties. These models are kinetically equivalent to a mobile carrier model.
120299434
Contributions to the theory of active transport: II. The gate type non-carrier mechanism and generalizations concerning tracer flow, efficiency, and measurement of energy expenditure
The gate type non-carrier mechanism, an active transport model, is discussed. In this mechanism, the actively transported particle passes through the gate itself by means of a series of reorganizations of the active transport mechanism. The net rate of transport, the rate of transport in either direction, and the efficiency of this model are analyzed. It is shown that on the basis of these analyses alone, this mechanism cannot be distinguished from a carrier mechanism. Three generalizations which apply to many individual type active transport models are then discussed. These pertain to (1) the dependency of the flow in one direction on the cencentration of the particles on the opposite side of the membrane, (2) the possibility of very high efficiencies for these models independent of the rate of the active transport, and (3) the methods whereby the energy expended in the active transport may be experimentally found.
27413429
Selected titles
Genetic Toxicology of Environmental Chemicals Edited by Claes Ramel, Bo Lambert and Jan Magnusson Alan R. Liss, 1986 Guidelines on the Use of Mutagenicity Tests in the Toxicological Evaluation of Chemicals A Report of the DNH and W/DOE Environmental Contaminants Advisory Committee on Mutagenesis Published by Authority of the Minister of National Health and Welfare and the Minister of the Environment, Ontario, 1986
9943078
Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells
SummaryThe transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate substites are required for the preferred substrates. Cl− and HCO3−, to account for tight binding in the transition state (Kdiss≃1μm). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unsual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and “tunneling”, which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.
23912133
Coupling mechanisms in active transport.
In primary and secondary active transport, the mobility and specificity of the carrier are controlled, over the course of the transport reaction, in accordance with a set of 'rules'. The rules are shown to depend on two mechanisms: a substrate--either the driving substrate (a transported ion or ATP) or the driven substrate--may shift a conformational equilibrium or accelerate a rate-limiting conformational change. From an analysis of coupling mechanisms the following conclusions emerge. (i) The ratio of coupled to uncoupled flux, which should be large, cannot be greater than the ratio of substrate dissociation constants in an initial complex and a conformationally altered state. A minimum value for the increased binding force can be estimated from steady-state constants. (ii) In an ordered mechanism, slippage is expected at high concentrations of the substrate adding to the carrier second, while slippage of the first substrate should remain low. (iii) Slippage in coupled transport is minimized if the driven substrate is last on in loading the carrier and last off in unloading, while the reverse order makes the affinity high in loading and low in unloading, as required for efficient transfer from one compartment to another; hence the preferred mechanism may depend on prevailing physiological conditions. (iv) A coupled transport system can be transformed into a facilitated system for one substrate or both if the control of carrier mobility is undermined through modification of the carrier.
93877710
Enzyme structure and mechanism
This is the second edition of this biological reference aimed at undergraduates and graduates. The book covers the structure and mechanism of enzymes, creating a guide to the current understanding of enzymology.
12992759
Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis.
Carrier-linked transport through biomembranes is treated under the view of catalysis. As in enzymes, substrate-protein interaction yields catalytic energy in overcoming the activation barrier. At variance with enzymes, catalytic energy is concentrated on structural changes of the carrier rather than on the substrate destabilization for facilitating the global protein rearrangements during transport. A transition state is invoked in which the binding site assumes the best fit to the substrate, whereas in the two ground (internal and external) states, the fit is poor. The maximum binding energy released in the transition state provides catalytic energy to enable the large carrier protein transformations associated with transport. This "induced transition fit" (ITF) of carrier catalysis provides a framework of rules, concerning specificity, unidirectional versus exchange type transport, directing inhibitors to the ground state instead of the transition state, and excluding simultaneous chemical and transport catalysis (vectorial group translocation). The possible role of external energy sources (ATP and Deltapsi) in supplementing the catalytic energy is elucidated. The analysis of the structure-function relationship based on new carrier structures may be challenged to account for the workings of the ITF.
24390494
Transport catalysis.
Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This "induced transition fit" (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for "low affinity" transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.
36780145
Utilization of binding energy and coupling rules for active transport and other coupled vectorial processes.
Publisher Summary This chapter describes the utilization of binding energy and coupling rules for active transport and other coupled vectorial processes. The primary role of binding energy is kinetic. Binding energy is utilized to avoid high- or low-energy intermediates along the reaction path under physiological conditions. High-energy intermediates are likely to introduce a barrier to rapid turnover of the system, while low-energy intermediates cause the enzyme to fall into an energy well from which escape is difficult. The energies of the intermediates in the reaction cycle of the Ca 2+ -ATPase from sarcoplasmic reticulum illustrate how the energies are balanced in a system of this kind to avoid high- or low-energy intermediates. The utilization of binding energies to avoid high- or low-energy intermediates is described by interaction energies. The coupling process is described by a set of rules. These rules generally represent enzyme specificities for catalysis that change in different states of the enzyme.
13585767
Ins and outs of major facilitator superfamily antiporters.
The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.
4310684
Unlocking the molecular secrets of sodium-coupled transporters
Transmembrane sodium-ion gradients provide energy that can be harnessed by 'secondary transporters' to drive the translocation of solute molecules into a cell. Decades of study have shown that such sodium-coupled transporters are involved in many physiological processes, making them targets for the treatment of numerous diseases. Within the past year, crystal structures of several sodium-coupled transporters from different families have been reported, showing a remarkable structural conservation between functionally unrelated transporters. These atomic-resolution structures are revealing the mechanism of the sodium-coupled transport of solutes across cellular membranes.
23606121
The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters.
Crystal structures of the bacterial amino acid transporter LeuT have provided the basis for understanding the conformational changes associated with substrate translocation by a multitude of transport proteins with the same fold. Biochemical and modeling studies led to a "rocking bundle" mechanism for LeuT that was validated by subsequent transporter structures. These advances suggest how coupled solute transport might be defined by the internal symmetry of proteins containing inverted structural repeats.
42176592
Structure and function of Na(+)-symporters with inverted repeats.
Symporters are membrane proteins that couple energy stored in electrochemical potential gradients to drive the cotransport of molecules and ions into cells. Traditionally, proteins are classified into gene families based on sequence homology and functional properties, for example the sodium glucose (SLC5 or Sodium Solute Symporter Family, SSS or SSF) and GABA (SLC6 or Neurotransmitter Sodium Symporter Family, NSS or SNF) symporter families [1-4]. Recently, it has been established that four Na(+)-symporter proteins with unrelated sequences have a common structural core containing an inverted repeat of 5 transmembrane (TM) helices [5(**)-8(**)]. Analysis of these four structures reveals that they reside in different conformations along the transport cycle providing atomic insight into the mechanism of sodium solute cotransport.
206525138
Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1
Triangulating to Mechanism Cellular uptake and release of a variety of substrates are mediated by secondary transporters, but no crystal structures are known for all three fundamental states of the transport cycle, which has limited explanations for their proposed mechanisms. Shimamura et al. (p. 470) report a 3.8-angstrom structure of the inward-facing conformation of the bacterial sodium-benzylhydantoin transport protein, Mhp1, complementing the other two available structures. Molecular modeling for the interconversions of these structures shows a simple rigid body rotation of four helices relative to the rest of the structure in which the protein switches reversibly from outward- to inward-facing. Three complementary crystal structures reveal the mechanism of a transport protein in molecular dynamics simulations. The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
26227077
Structural perspectives on secondary active transporters.
Secondary active transporters catalyze the concentrative transport of substrates across lipid membranes by harnessing the energy of electrochemical ion gradients. These transporters bind their ligands on one side of the membrane, and undergo a global conformational change to release them on the other side of the membrane. Over the last few years, crystal structures have captured several bacterial secondary transporters in different states along their transport cycle, providing insight into possible molecular mechanisms. In this review, we summarize recent findings focusing on the emerging structural and mechanistic similarities between evolutionary diverse transporters. We also discuss the structural basis of substrate binding, ion coupling and inhibition viewed from the perspective of these similarities.
29962915
Mechanism of free energy coupling in active transport.
PERSPECTIVES AND SUMMARy 379 OVERALL REACTION AND THERMODyNAMICS 381 Free Energy Bookkeeping 383 Questions oJ Stoichiometry 385 Closeness to Equilibrium 386 PATHWAY FOR ION TRANSLOCATION 386 Alternating Access M odel 386 Access Channels 389 How Can the Protein Change the Chemical Potential? 391 SEPARATE PATHWAY FOR ATP PROCESSING 395 Pumps with Phosphoenzyme Intermediates 396 FoFI Systems 397 COUPLING BETWEEN SEPARATE PATHWAyS 398 ATP-Linked Transport 398 Counter-Transport Systems 400 CONFLUENT PATHWAyS 402 MONOMER OR OLIGOMER 403 CONCLUSION 404
84266087
The ADP,ATP shuttle of the mitochondrion
Abstract The most active membrane transport system in eukaryotic cells — the ADP,ATP counter-exchange unique to mitochondria — is characterized by its transport activity, its coupling to energy transfer and its molecular properties. The study of this system has advanced our knowledge of the mechanism of a membrane carrier, and has generated the ‘gated pore' model.
8069157
Expression of substrate specificity in facilitated transport systems
SummaryIn facilitated transport systems the carrier reorientation step is shown to be largely independent of the forces of interaction between the substrate and the carrier site, whereas in coupled systems (obligatory exchange or cotransport) reorientation proceeds at the expense of the binding force developed in the transition state. In consequence, the expression of substrate specificity is expected to differ in the two systems. In the facilitated transport of analogs no larger than the normal substrate, the affinity but not the maximum rate of transport can vary widely; with larger analogs, both the affinity and rale can vary if steric constraints are more severe in the translocation step than in binding. In coupled transport, by contrast, the translocation step can be highly sensitive to the structure of the substrate, and binding much less sensitive. The theory agrees with published observations on facilitated systems for choline and glucose in erythrocytes, as well as on Na+-coupled systems for the same substrates in other cells. The following mechanism, which could account for the behavior, is proposed. In facilitated systems, the transport site fits the substrate closely and retains its shape as the carrier undergoes reorientation. In coupled systems, the site is initially looser, but during carrier reorientation it contracts around the substrate. In both systems, the carrier encloses the substrate during the translocation step, though for a different reason: in coupled but not in facilitated systems the binding force enormously increases in the enclosed state, through a chelation effect. In both systems, steric interference with enclosure retards the translocation of bulky substrate analogs.
238824
The molecular logic of sodium-coupled neurotransmitter transporters
Synaptic transmission at chemical synapses requires the removal of neurotransmitter from extracellular spaces. At synapses in the central nervous system, this is accomplished by sodium-coupled transport proteins, integral membrane proteins that thermodynamically couple the uptake of neurotransmitter to the uptake of sodium and, in some cases, the uptake and export of additional ions. Recent X-ray crystallographic studies have revealed the architecture of the two major families of neurotransmitter transporters and, together with additional biochemical and biophysical studies, have provided insights into mechanisms of ion coupling, substrate uptake, and inhibition of transport.
7108705
Lessons from lactose permease.
An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H+ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H+ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H+ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H+-binding sites to either side of the membrane.
30237208
Structural and mechanistic diversity of secondary transporters.
Recent reports on the three-dimensional structure of secondary transporters have dramatically increased our knowledge of the translocation mechanism of ions and solutes. The structures of five transporters at atomic resolution have yielded four different folds and as many different translocation mechanisms. The structure of the glutamate transporter homologue GltPh confirmed the role of pore-loop structures as essential parts of the translocation mechanism in one family of secondary transporters. Biochemical evidence for pore-loop structures in several other families suggest that they might be common in secondary transporters, adding to the structural and mechanistic diversity of secondary transporters.
7749253
NhaA crystal structure: functional–structural insights
SUMMARY Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic membrane and many intracellular membranes. They are essential for Na+, pH and volume homeostasis, which are crucial processes for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt-resistance in plants. Many Na+/H+ antiporters are tightly regulated by pH. Escherichia coli NhaA Na+/H+ antiporter, a prototype pH-regulated antiporter, exchanges 2 H+ for 1 Na+ (or Li+). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, `pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na+/H+ exchanger (NHE1) was constructed, paving the way to a rational drug design.
39648413
Nucleotide exchange in mitochondria: insight at a molecular level.
Mitochondrial carrier proteins are embedded in the inner mitochondrial membrane and ensure the transport of many important metabolites. The ADP/ATP carrier imports ADP into the mitochondrial matrix in exchange for ATP after synthesis. It is the most studied mitochondrial carrier and its structure was the first to be unraveled at high resolution. The structure reveals six transmembrane helices forming a tightly closed bundle toward the matrix and a funnel-shaped cavity opening toward the intermembrane space. The cavity ends in a narrow pit 10A from the matrix. The analysis of residues located in the cavity hints at the mechanism of binding of adenine nucleotides. Additionally, the presence of conserved proline residues in three sharply kinked helices suggests a translocation mechanism.
10125033
The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily.
The major facilitator superfamily represents the largest group of secondary active membrane transporters in the cell. The 3.3A resolution structure of a member of this protein superfamily, the glycerol-3-phosphate transporter from the Escherichia coli inner membrane, reveals two domains connected by a long central loop. These N- and C-terminal domains, each containing a six-helix bundle, are related by pseudo-twofold symmetry. A substrate translocation pore is located between the two domains and is open to the cytoplasm. Two arginines at the closed end of the pore comprise the substrate-binding site. Biochemical experiments show that, upon substrate binding, the protein adopts a more compact conformation. The crystal structure suggests that the transporter operates through a single binding site, alternating access mechanism via a rocker-switch type of movement of the N- and C-terminal domains. The structure and mechanism of the glycerol-3-phosphate transporter form a paradigm for other members of the major facilitator superfamily.
40180297
Lactose permease as a paradigm for membrane transport proteins (Review)
Our structural knowledge of the major facilitator superfamily (MFS) has dramatically increased in the past year with three structures of proteins from the MFS (oxalate/formate antiporter; lactose/proton symporter and the Pi/glycerol-3-phosphate antiporter). All three structures revealed 12 transmembrane helices forming two distinct domains and could imply that members of the MFS have preserved both secondary as well as tertiary structural elements during evolution. Lactose permease, a particularly well-studied member of the MFS, has been extensively explored by a number of molecular biological, biochemical and biophysical approaches. In this review, we take a closer look at the structure of LacY and incorporate a wealth of biochemical and biophysical data in order to propose a possible mechanism for lactose/proton symport. In addition, we make some brief comparisons between the structures of LacY and GlpT.
4338748
Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside
ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 Å by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six α-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a ‘pit’ to a ‘channel’ conformation.
1244746
Mitochondrial carriers function as monomers.
Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35 years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.
4694758
Wanderings in bioenergetics and biomembranes.
Having worked for 55 years in the center and at the fringe of bioenergetics, my major research stations are reviewed in the following wanderings: from microsomes to mitochondria, from NAD to CoQ, from reversed electron transport to reversed oxidative phosphorylation, from mitochondrial hydrogen transfer to phosphate transfer pathways, from endogenous nucleotides to mitochondrial compartmentation, from transport to mechanism, from carrier to structure, from coupling by AAC to uncoupling by UCP, and from specific to general transport laws. These wanderings are recalled with varying emphasis paid to the covered science stations.
30874107
Structural basis for lipid‐mediated interactions between mitochondrial ADP/ATP carrier monomers
The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein–protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein–protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.
25601065
Highly conserved charge-pair networks in the mitochondrial carrier family.
Selection for regain-of-function mutations in the yeast ADP/ATP carrier AAC2 has revealed an unexpected series of charge-pairs. Four of the six amino acids involved are found in the mitochondrial energy transfer motifs used to define this family of proteins. As such, the results found with the ADP/ATP carrier may apply to the family as a whole. Mitochondrial carriers are built from three homologous domains, each with the conserved motif PX(D,E)XX(K,R). Neutralization of the conserved positive charges at K48, R152 or R252 in these motifs results in respiration defective yeast. Neutralization of the negative charges at D149 and D249 also make respiration defective yeast, though E45G or E45Q mutants are able to grow on glycerol. Regain of function occurs when a complementary charge is lost from another site in the molecule. This phenomenon has been observed independently eight times and thus is strong evidence for charge-pairs existing between the affected residues. Five different charge-pairs have been detected in the yeast AAC2 by this method and three more can be predicted based on homology between the domains. The highly conserved charge-pairs occurring within or between the three mitochondrial energy transfer signatures seem to be a critical feature of mitochondrial carrier structure, independent of the substrates transported. Conformational switching between alternative charge-pairs may constitute part of the basis for transport.
44424094
The mechanism of transport by mitochondrial carriers based on analysis of symmetry
The structures of mitochondrial transporters and uncoupling proteins are 3-fold pseudosymmetrical, but their substrates and coupling ions are not. Thus, deviations from symmetry are to be expected in the substrate and ion-binding sites in the central aqueous cavity. By analyzing the 3-fold pseudosymmetrical repeats from which their sequences are made, conserved asymmetric residues were found to cluster in a region of the central cavity identified previously as the common substrate-binding site. Conserved symmetrical residues required for the transport mechanism were found at the water–membrane interfaces, and they include the three PX[DE]XX[RK] motifs, which form a salt bridge network on the matrix side of the cavity when the substrate-binding site is open to the mitochondrial intermembrane space. Symmetrical residues in three [FY][DE]XX[RK] motifs are on the cytoplasmic side of the cavity and could form a salt bridge network when the substrate-binding site is accessible from the mitochondrial matrix. It is proposed that the opening and closing of the carrier may be coupled to the disruption and formation of the 2 salt bridge networks via a 3-fold rotary twist induced by substrate binding. The interaction energies of the networks allow members of the transporter family to be classified as strict exchangers or uniporters.
1956116
The conserved substrate binding site of mitochondrial carriers.
Mitochondrial carriers transport nucleotides, co-factors and metabolic intermediates across the inner mitochondrial membrane permeability barrier. They belong to a family of transporters unique to eukaryotes and they differ in structure and transport mechanism from other secondary transporters. The main structural fold consists of a barrel of six transmembrane alpha-helices closed at the matrix side by a salt-bridge network at the bottom of the cavity. The significant sequence conservation in the mitochondrial carrier family suggests that specific recognition of substrates is coupled to a common mechanism of transport. We have identified a common substrate binding site comprising residues that are highly conserved and, as demonstrated by mutagenesis, are essential for function. The binding site explains substrate selectivity, ion coupling and the effects of the membrane potential on transport. The main contact points in the site are related by threefold symmetry like the common structural fold. The substrate is bound at the midpoint of the membrane and may function as a pivot point for the movements of the transmembrane alpha-helices as the carrier changes conformation. The trigger for the translocation event is likely to be the substrate-induced perturbation of the salt bridge network at the bottom of the cavity.
6729920
Electrostatic funneling of substrate in mitochondrial inner membrane carriers
Exchange of ATP and ADP across mitochondrial membrane replenishes the cytoplasm with newly synthesized ATP and provides the mitochondria with the substrate ADP for oxidative phosphorylation. The sole means of this exchange is the mitochondrial ADP/ATP carrier (AAC), a membrane protein that is suggested to cycle between two conformationally distinct states, cytosolic-open (c-state) and matrix-open (m-state), thereby shuttling nucleotides across the inner mitochondrial membrane. However, the c-state is the only structurally resolved state, and the binding site of ADP remains elusive. Here, we present ≈0.3 μs of all-atom MD simulations of the c-state revealing rapid, spontaneous binding of ADP to deeply positioned binding sites within the AAC lumen. To our knowledge, a complete ligand-binding event has heretofore not been described in full atomic detail in unbiased simulations. The identified ADP-bound state and additional simulations shed light on key structural elements and the initial steps involved in conversion to the m-state. Electrostatic analysis of trajectories reveals the presence of an unusually strong positive electrostatic potential in the lumen of AAC that appears to be the main driving force for the observed spontaneous binding of ADP. We provide evidence that the positive electrostatic potential is likely a common attribute among the entire family of mitochondrial carriers. In addition to playing a key role in substrate recruitment and translocation, the electropositivity of mitochondrial carriers might also be critical for their binding to the negatively charged environment of the inner mitochondrial membrane.
23976592
Mitochondrial carriers in the cytoplasmic state have a common substrate binding site.
Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP/ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted.
35726914
Mitochondrial carrier proteins
Ten mitochondrial carriers have been purified from animal mitochondria. They are small proteins with a molecular mass ranging from 28 to 34 kDa on SDS‐PAGE. So far, five of these proteins have been sequenced. Their polypeptide chain consists of three tandemly related sequences of about 100 amino acids. The repeats of the different proteins are related and probably fold into two transmembrane a‐helices linked by an extra‐membrane loop. The features of this family are also present in several proteins of unknown function characterized by DNA sequencing. Isoforms of some carriers have been found. All mitochondrial carriers investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport. The only exception is the carnitine carrier that proceeds via a ping‐pong mechanism. Three mitochondrial carriers have been expressed in yeast and two overexpressed in E. coli and refolded in active form.
22345730
Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel.
The ADP/ATP carrier (AAC) is a membrane protein of paramount importance for the energy-fueling function of the mitochondria, transporting ADP from the intermembrane space to the matrix and ATP in the opposite direction. On the basis of the high-resolution, 2.2-A structure of the bovine carrier, a total of 0.53 micros of classical molecular dynamics simulations were conducted in a realistic membrane environment to decipher the early events of ADP (3-) translocation across the inner membrane of the mitochondria. Examination of apo-AAC underscores the impermeable nature of the carrier, impeding passive transport of permeants toward the matrix. The electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential forms a privileged passageway anticipated to drive the diphosphate nucleotide rapidly toward the bottom of the internal cavity. This conjecture is verified in the light of repeated, independent numerical experiments, whereby the permeant is dropped near the mouth of the mitochondrial carrier. Systematic association of ADP (3-) to the crevice of the AAC, an early event in its transport across the inner membrane, is accompanied by the formation of an intricate network of noncovalent bonds. Simulations relying on the use of an adaptive biasing force reveal for the first time that the proposed binding site corresponds to a minimum of the free energy landscape delineating the translocation of ADP (3-) in the carrier. The present work paves the way to the design of novel nucleotides and new experiments aimed at unveiling key structural features in the chronology of ADP/ATP transport across the mitochondrial membrane.
9545480
Structural determination of wild-type lactose permease
Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H+ binding. To initiate transport, binding of sugar and/or an H+ electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
1501987
Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY
Cation‐coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand‐free X‐ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation. No sugar‐binding site is observed in the absence of ligand, and deprotonation of the key residue Glu269 is associated with ligand binding. Thus, substrate induces formation of the sugar‐binding site, as well as the initial step in H+ transduction.
36908983
Structure and Mechanism of the Lactose Permease of Escherichia coli
Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, β-D-galactopyranosyl-1-thio-β-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.
205222401
Structure of a fucose transporter in an outward-open conformation
The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.
11520791
The Cys154-->Gly mutation in LacY causes constitutive opening of the hydrophilic periplasmic pathway.
The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein, while the Cys154-->Gly mutant is crippled conformationally. The mutant binds sugar with high affinity, but catalyzes very little translocation across the membrane. In order to further investigate the defect in the mutant, fluorescent maleimides were used to examine the accessibility/reactivity of single-Cys LacY in right-side-out membrane vesicles. As shown previously, sugar binding induces an increase in reactivity of single-Cys replacements in the tightly packed periplasmic domain of wild-type LacY, while decreased reactivity is observed on the cytoplasmic side. Thus, the predominant population of wild-type LacY in the membrane is in an inward-facing conformation in the absence of sugar, sugar binding induces opening of a hydrophilic pathway on the periplasmic side, and the sugar-binding site is alternatively accessible to either side of the membrane. In striking contrast, the accessibility/reactivity of periplasmic Cys replacements in the Cys154-->Gly background is very high in the absence of sugar, and sugar binding has little or no effect. The observations indicate that an open hydrophilic pathway is present on the periplasmic side of the Cys154-->Gly mutant and that this pathway is unaffected by ligand binding, a conclusion consistent with findings obtained from single-molecule fluorescence and double electron-electron resonance.
32677357
A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability.
Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.
6777217
Sugar binding and protein conformational changes in lactose permease.
Lactose permease is an integral membrane protein that uses the cell membrane's proton gradient for import of lactose. Based on extensive biochemical data and a substrate-bound crystal structure, intermediates involved in lactose/H(+) co-transport have been suggested. Yet, the transport mechanism, especially the coupling of protonation states of essential residues and protein conformational changes involved in the transport, is not understood. Here we report molecular-dynamics simulations of membrane-embedded lactose permease in different protonation states, both in the presence and in the absence of lactose. The results analyzed in terms of pore diameter, salt-bridge formation, and substrate motion, strongly implicate Glu(269) as one of the main proton translocation sites, whose protonation state controls several key steps of the transport process. A critical ion pair (Glu(269) and Arg(144)) was found to keep the cytoplasmic entrance open, but via a different mechanism than the currently accepted model. After protonation of Glu(269), the salt bridge between Glu(269) and Arg(144) was found to break, and Arg(144) to move away from Glu(269), establishing a new salt bridge with Glu(126); furthermore, neutralization of Glu(269) and the displacement of Arg(144) and consequently of water molecules from the interdomain region was seen to initiate the closing of the cytoplasmic half channel (2.6-4.0 A reduction in diameter in the cytoplasmic constriction region in 10 ns) by allowing hydrophobic surfaces of the N- and C-domains to fuse. Charged Glu(269) was found to strongly bind the lactose permeant, indicating that proton transfer from water or another residue to Glu(269) is a prerequisite for unbinding of lactose from the binding pocket.
13785210
Sugar binding in lactose permease: anomeric state of a disaccharide influences binding structure.
Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for alpha-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with beta(alpha)-d-galactopyranosyl-(1,1)-beta-d-galactopyranoside (betabeta-(Galp)(2)) and alphabeta-(Galp)(2) result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and alphabeta-(Galp)(2) has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for alphabeta-(Galp)(2) than betabeta-(Galp)(2), i.e. -10.7(+/-0.7) and -3.1(+/-1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with betabeta-(Galp)(2) binding resulting in larger distortion than alphabeta-(Galp)(2).
7248404
Conformational change in an MFS protein: MD simulations of LacY.
Molecular dynamics simulations of lactose permease (LacY) in a phospholipid bilayer reveal the conformational dynamics of the protein. In inhibitor-bound simulations (i.e., those closest to the X-ray structure) the protein was stable, showing little conformational change over a 50 ns timescale. Movement of the bound inhibitor, TDG, to an alternative binding mode was observed, so that it interacted predominantly with the N-terminal domain and with residue E269 from the C-terminal domain. In multiple ligand-free simulations, a degree of domain closure occurred. This switched LacY to a state with a central cavity closed at both the intracellular and periplasmic ends. This may resemble a possible intermediate in the transport mechanism. Domain closure occurs by a combination of rigid-body movements of domains and of intradomain motions of helices, especially TM4, TM5, TM10, and TM11. A degree of intrahelix flexibility appears to be important in the conformational change.
9927365
Structure and transport mechanism of the bacterial oxalate transporter OxlT.
Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.
12849456
Sugar binding induces the same global conformational change in purified LacY as in the native bacterial membrane
Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H+ symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H+-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.
12081912
Residues gating the periplasmic pathway of LacY.
X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H(+) translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H(+)-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H(+) symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated ( approximately 29 A), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40-->Cys/Asn245-->Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.
24629761
Clogging the periplasmic pathway in LacY.
The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein. Crystal structures of wild-type and mutant LacY all exhibit an inward-facing conformation with an open cytoplasmic pathway and a tightly packed periplasmic side, which makes the binding site inaccessible from the outside. However, biochemical and biophysical findings provide strong evidence that occupation of the sugar-binding site leads to an increased probability of opening of a hydrophilic pathway on the periplasmic side and closing of the cytoplasmic cavity. By this means, the sugar-binding site becomes accessible to either side of the membrane in alternating fashion. To extend studies on the relationship between the periplasmic pathway and transport activity, engineered single-Cys replacements in the periplasmic pathway were reacted to completion with thiol reagents, and the effects on transport and sugar binding were tested. Inactivation correlates for the most part with the size of the modifying reagent, although the position of the Cys replacement is also important. However, sugar binding is unaffected. The results suggest that placement of a relatively large moiety in the putative periplasmic cleft of LacY likely prevents closure, an essential step in the transport cycle, without significantly altering access of sugar to the binding site.
31093418
Helix dynamics in LacY: helices II and IV.
Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H+ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within approximately 4.2 A of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.
19973056
Site-directed alkylation of LacY: effect of the proton electrochemical gradient.
Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H(+) electrochemical gradient (Delta(mu (H)+), interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Delta(mu (H)+), like sugar, acts to increase the probability of opening on the periplasmic side of LacY.
23317464
Site-directed alkylation and the alternating access model for LacY
In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant alkylating agent N-ethylmaleimide in right-side-out membrane vesicles. Here we present the results in the context of the crystal structure of LacY. Engineered Cys replacements located near or within the inward-facing hydrophilic cavity or at other solvent-accessible positions in LacY react well with this alkylating agent. Cys residues facing the low dielectric of the membrane or located in tightly packed regions of the structure react poorly. Remarkably, in the presence of ligand, increased reactivity is observed with Cys replacements located predominantly on the periplasmic side of the sugar-binding site. In contrast, other Cys replacements largely on the cytoplasmic side of the binding site exhibit decreased reactivity. Furthermore, both sets of Cys replacements in the putative cavities are located at the periplasmic (increased reactivity) and cytoplasmic (decreased reactivity) ends of the same helices and distributed in a pseudosymmetrical manner. The results are consistent with a model in which the single sugar-binding site in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities.
14659710
Single-molecule FRET reveals sugar-induced conformational dynamics in LacY
The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H+ symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.
20191816
Direct sugar binding to LacY measured by resonance energy transfer.
Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Förster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the Förster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.
23121195
Sugar binding induces an outward facing conformation of LacY
According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H+ symport, a cavity facing the periplasmic side is thought to open with closure of the inward-facing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electron–electron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxide-labeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-154→Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 Å. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 Å. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-154→Gly mutant, sugar-induced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.
14907453
Probing of the rates of alternating access in LacY with Trp fluorescence
Sugar/H+ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H+ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pKa of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s−1. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pKa = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s−1. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.
371319
Residues in the H+ translocation site define the pKa for sugar binding to LacY.
A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.
5272017
Protonation and sugar binding to LacY
The effect of bulk-phase pH on the apparent affinity (Kdapp) of purified wild-type lactose permease (LacY) for sugars was studied. Kdapp values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. Kdapp for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pKa of ≈10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H+ symport in either direction across the membrane.
31276325
Opening and closing of the periplasmic gate in lactose permease
X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H+ translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants—Ile-40 → Cys/Asn-245 → Cys, Thr-45 → Cys/Asn-245 → Cys, and Ile-32 → Cys/Asn-245 → Cys—located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than ≈15 Å in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than ≈15 Å in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.
245787
Simulation of spontaneous substrate binding revealing the binding pathway and mechanism and initial conformational response of GlpT.
Glycerol 3-phosphate transporter (GlpT) mediates the import of glycerol 3-phosphate (G3P) using the gradient of inorganic phosphate (P(i)). To study the process and mechanism of substrate binding and to investigate the protein's initial response, we performed equilibrium simulations of wild-type GlpT and several of its mutant forms in membranes in the presence of all physiologically relevant substrates (P(i)(-), P(i)(2-), G3P(-), and G3P(2-)). The simulations capture spontaneous substrate binding of GlpT, driven by the positive electrostatic potential of the lumen. K80 is found to act as a "hook" making the first encounter with the substrate and guiding it toward the binding site, where it binds tightly to R45, a key binding site residue that acts as a "fork" holding the substrate. R269 establishes no direct contact with the substrate during the simulations, a surprising behavior given its structural pseudosymmetry to R45. In all substrate-bound systems, partial closing of the cytoplasmic half of GlpT was observed. The substrate appears to stabilize the partially occluded state, as in the two apo simulations either no closing was observed or the protein reverted to its open form toward the end of the simulation, whereas in all substrate-bound systems, a stable partially closed state was produced. Along with the modulation of the periplasmic salt bridge network, these substrate-induced events destabilize the periplasmic half while inducing a closure in the cytoplasmic half, thus capturing the early stages of the proposed rocker-switch mechanism in GlpT.
4368184
Structure of a glutamate transporter homologue from Pyrococcus horikoshii
Glutamate transporters are integral membrane proteins that catalyse the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.
4420420
Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter
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 GltPh, a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-β-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 α-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.
4415744
Transport mechanism of a bacterial homologue of glutamate transporters
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, GltPh, which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of GltPh in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which GltPh and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.
29282763
Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats
Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 Å apart in the crystal structures of GltPh, but ≈10 Å apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue d,l-threo-β-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.
14689598
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.
34610845
Fluorometric measurements of conformational changes in glutamate transporters.
Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na(+)-dependent conformational changes preceding glutamate binding. We furthermore show that Na(+) and H(+) are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.
4420334
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.
206514093
The Crystal Structure of a Sodium Galactose Transporter Reveals Mechanistic Insights into Na+/Sugar Symport
Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ∼3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.
205523029
Crystal structure of the carnitine transporter and insights into the antiport mechanism
CaiT is a membrane antiporter that catalyzes the exchange of l-carnitine with γ-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 Å. We crystallized CaiT as a homotrimer complex, in which each protomer contained 12 transmembrane helices and 4 l-carnitine molecules outlining the transport pathway across the membrane. Mutagenesis studies revealed a primary binding site at the center of the protein and a secondary substrate-binding site at the bottom of the intracellular vestibule. These results, together with the insights obtained from structural comparison with structurally homologous transporters, provide mechanistic insights into the association between substrate translocation and the conformational changes of CaiT.
25291541
Structure and Mechanism of a Na+-Independent Amino Acid Transporter
APC Transporter Structure Amino acid, polyamine, and organocation (APC) transporters that move a range of organic molecules across the cell membrane are important in many cellular processes. Shaffer et al. (p. 1010; published online 16 July) report the crystal structure of apoApcT, a proton-dependent APC transporter. The structure shows similarity to the sodium-coupled amino acid transporter LeuT and has an amino group from a lysine in an equivalent position to a sodium ion in LeuT. This suggests common principles between proton- and sodium-coupled transporters. The structure of the transporter ApcT reveals common architectural principles between proton- and sodium-coupled transporters. Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.
4356016
Structure of a Prokaryotic Virtual Proton Pump at 3.2 Å Resolution
To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5–4) by mounting extreme acid-resistance responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a ‘virtual proton pump’ in the inner membrane, called AdiC, that imports l-arginine from the gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic β-cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 Å resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.
205219597
Mechanism of substrate recognition and transport by an amino acid antiporter
In extremely acidic environments, enteric bacteria such as Escherichia coli rely on the amino acid antiporter AdiC to expel protons by exchanging intracellular agmatine (Agm2+) for extracellular arginine (Arg+). AdiC is a representative member of the amino acid-polyamine-organocation (APC) superfamily of membrane transporters. The structure of substrate-free AdiC revealed a homodimeric assembly, with each protomer containing 12 transmembrane segments and existing in an outward-open conformation. The overall folding of AdiC is similar to that of the Na+-coupled symporters. Despite these advances, it remains unclear how the substrate (arginine or agmatine) is recognized and transported by AdiC. Here we report the crystal structure of an E. coli AdiC variant bound to Arg at 3.0 Å resolution. The positively charged Arg is enclosed in an acidic binding chamber, with the head groups of Arg hydrogen-bonded to main chain atoms of AdiC and the aliphatic portion of Arg stacked by hydrophobic side chains of highly conserved residues. Arg binding induces pronounced structural rearrangement in transmembrane helix 6 (TM6) and, to a lesser extent, TM2 and TM10, resulting in an occluded conformation. Structural analysis identified three potential gates, involving four aromatic residues and Glu 208, which may work in concert to differentially regulate the upload and release of Arg and Agm.
40949323
Discontinuous membrane helices in transport proteins and their correlation with function.
Alpha-helical bundles and beta-barrel proteins represent the two basic types of architecture known for integral membrane proteins. Irregular structural motifs have been revealed with the growing number of structures determined. "Discontinuous" helices are present in membrane proteins that actively transport ions. In the Ca(2+)-ATPase, a primary active transporter, and in the secondary transporters NhaA, LeuT(Aa), ClC H(+)/Cl(-) exchanger and Glt(Ph), the helical structure of two membrane segments is interrupted and the interjacent polypeptide chain forms an extended peptide. The discontinuous helices are integrated in the membrane either as transmembrane-spanning or hairpin-type segments. In addition, the secondary transporters have inverted internal duplication domains, which are only weakly correlated with their amino acid sequence. The symmetry comprises either parts of or the complete molecule, but always includes the discontinuous helices. The helix-peptide-helix motif is correlated with the ion translocation function. The extended peptides with their backbone atoms, the helix termini and the polar/charged amino acid residues in close vicinity provide the basis for ion recognition, binding and translocation.
37572006
Structure and Mechanism of an Amino Acid Antiporter
Antiporter Antics Bacteria that survive in the acidic environment of the stomach have mechanisms to maintain a high intracellular pH. In Escherichia coli, glutamate (Glu) and arginine (Arg) are decarboxylated intracellularly and the reaction products are exchanged with extracellular Glu and Arg. Gao et al. (p. 1565, published online 28 May) now report a crystal structure of AdiC, an arginine:agmatine antiporter from E. coli. AdiC exhibits the same fold as that of the Na+-coupled symporters, including LeuT. It contains 12 transmembrane segments, forms a homodimer, and exists in an outward-facing, open conformation in the crystals. The structure, together with biochemical data, suggests how the antiporter senses the pH and responds to transport the reaction product agmatine out of the cell and Arg into the cell. The architecture of a membrane protein channel helps explain how Escherichia coli survive the acidic conditions of the stomach. Virulent enteric pathogens such as Escherichia coli strain O157:H7 rely on acid-resistance (AR) systems to survive the acidic environment in the stomach. A major component of AR is an arginine-dependent arginine:agmatine antiporter that expels intracellular protons. Here, we report the crystal structure of AdiC, the arginine:agmatine antiporter from E. coli O157:H7 and a member of the amino acid/polyamine/organocation (APC) superfamily of transporters at 3.6 Å resolution. The overall fold is similar to that of several Na+-coupled symporters. AdiC contains 12 transmembrane segments, forms a homodimer, and exists in an outward-facing, open conformation in the crystals. A conserved, acidic pocket opens to the periplasm. Structural and biochemical analysis reveals the essential ligand-binding residues, defines the transport route, and suggests a conserved mechanism for the antiporter activity.
206516689
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.
4315958
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.
41229104
Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation
The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na+; later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule ≈11 Å above the bound leucine and 2 Na+. We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na+-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-β-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 Å the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na+-coupled symport.
26695747
LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake
Tricyclic antidepressants exert their pharmacological effect—inhibiting the reuptake of serotonin, norepinephrine, and dopamine—by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.
4472020
Antidepressant specificity of serotonin transporter suggested by three LeuT–SSRI structures
Sertraline and fluoxetine are selective serotonin re-uptake inhibitors (SSRIs) that are widely prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess halogen atoms at specific positions, which are key determinants for the drugs' specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT markedly reduces the transporter's affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only nonconserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the protein's HBP.
38438760
The mechanism of a neurotransmitter:sodium symporter--inward release of Na+ and substrate is triggered by substrate in a second binding site.
Eukaryotic neurotransmitter:sodium symporters (NSSs), targets for antidepressants and psychostimulants, terminate neurotransmission by sodium-driven reuptake. The crystal structure of LeuT(Aa), a prokaryotic NSS homolog, revealed an occluded state in which one leucine and two Na(+) ions are bound, but provided limited clues to the molecular mechanism of transport. Using steered molecular dynamics simulations, we explored the substrate translocation pathway of LeuT. We identified a second substrate binding site located in the extracellular vestibule comprised of residues shown recently to participate in binding tricyclic antidepressants. Binding and flux experiments showed that the two binding sites can be occupied simultaneously. The substrate in the secondary site allosterically triggers intracellular release of Na(+) and substrate from the primary site, thereby functioning as a "symport effector." Because tricyclic antidepressants bind differently to this secondary site, they do not promote substrate release from the primary site and thus act as symport uncouplers and inhibit transport.
4360313
Single-molecule dynamics of gating in a neurotransmitter transporter homolog
Neurotransmitter:Na+ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na+ gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.
1592205
Mechanism for alternating access in neurotransmitter transporters
Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1–5 and TMs 6–10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.
205385960
The enlightening encounter between structure and function in the NhaA Na+-H+ antiporter.
Na(+)-H(+) antiporters are integral membrane proteins that exchange Na(+) for H(+) across the cytoplasmic membrane and many intracellular membranes. They are essential for Na(+), pH, and volume homeostasis, which are processes crucial for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt resistance in plants. Many Na(+)-H(+) antiporters are tightly regulated by pH. Escherichia coli NhaA, a prototype pH-regulated antiporter, exchanges 2H(+) for 1Na(+) (or Li(+)). The NhaA crystal structure has provided insight into the pH-regulated mechanism of antiporter action and revealed transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found with variations in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation.
4372674
Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH
The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.
30745070
Mechanism of Na+/H+ Antiporting
Na+/H+ antiporters are central to cellular salt and pH homeostasis. The structure of Escherichia coli NhaA was recently determined, but its mechanisms of transport and pH regulation remain elusive. We performed molecular dynamics simulations of NhaA that, with existing experimental data, enabled us to propose an atomically detailed model of antiporter function. Three conserved aspartates are key to our proposed mechanism: Asp164 (D164) is the Na+-binding site, D163 controls the alternating accessibility of this binding site to the cytoplasm or periplasm, and D133 is crucial for pH regulation. Consistent with experimental stoichiometry, two protons are required to transport a single Na+ ion: D163 protonates to reveal the Na+-binding site to the periplasm, and subsequent protonation of D164 releases Na+. Additional mutagenesis experiments further validated the model.
20759528
The influence of protonation states on the dynamics of the NhaA antiporter from Escherichia coli.
The crystal structure of NhaA Na(+)/H(+) antiporter of Escherichia coli has provided a basis to explore the mechanism of Na(+) and H(+) exchange and its regulation by pH. However, the dynamics and nature of the pH-induced changes in the proteins remained unknown. Using molecular mechanics methods, we studied the dynamic behavior of the hydrogen-bonded network in NhaA on shifting the pH from 4 to 8. The helical regions preserved the general architecture of NhaA throughout the pH change. In contrast, large conformational drifts occurred at pH 8 in the loop regions, and an increased flexibility of helix IVp was observed on the pH shift. A remarkable pH-induced conformational reorganization was found: at acidic pH helix X is slightly curved, whereas at alkaline pH, it is kinked around residue Lys(300). The barrier that exists between the cytoplasmic and periplasmic funnels at low pH is removed, and the two funnels are bridged by hydrogen bonds between water molecules and residues located in the TMSs IV/XI assembly and helix X at alkaline pH. In the variant Gly(338)Ser that lost pH control, a hydrogen-bonded chain between Ser(338) and Lys(300) was found to block the pH-induced conformational reorganization of helix X.
1780032
X-ray structure of EmrE supports dual topology model
EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a “dual topology” model.
33036600
EmrE, a model for studying evolution and mechanism of ion-coupled transporters.
EmrE is a small (110 residues) SMR transporter from Escherichia coli that extrudes positively charged aromatic drugs in exchange for two protons, thus rendering bacteria resistant to a variety of toxic compounds. Due to its size, stability and retention of its function upon solubilization in detergent, EmrE provides a unique experimental paradigm for the biochemical and biophysical studies of membrane based ion-coupled transporters. In addition, EmrE has been in center stage in the past two years because it provides also a paradigm for the study of the evolution of membrane proteins. Controversy around this topic is still going on and some novel concepts are surfacing that may contribute to our understanding of evolution of topology of membrane proteins. Furthermore, based on the findings that the cell multidrug transporters interact functionally we introduce the concept of a cell Resistosome.
13592403
When biochemistry meets structural biology: the cautionary tale of EmrE.
When biochemistry meets structural biology a more complete understanding of the mechanism of biological macromolecules is usually achieved. Several high-resolution structures of ion-coupled transporters have enriched the understanding of mechanisms of substrate recognition, translocation and coupling of substrate fluxes. However, two X-ray structures of EmrE, the smallest ion-coupled multi-drug transporter, raised questions over the veracity of the structural model and represented a cautionary tale about the difficulty of determining the 3D structures of membrane proteins and the dangers of ignoring biochemical results. The 3D structures of EmrE have since been retracted because of faulty software, but the suggestion that the protomers in the dimer are in an antiparallel topological orientation sparked controversy that is still ongoing.
35038794
Quasi-symmetry in the cryo-EM structure of EmrE provides the key to modeling its transmembrane domain.
Small multidrug resistance (SMR) transporters contribute to bacterial resistance by coupling the efflux of a wide range of toxic aromatic cations, some of which are commonly used as antibiotics and antiseptics, to proton influx. EmrE is a prototypical small multidrug resistance transporter comprising four transmembrane segments (M1-M4) that forms dimers. It was suggested recently that EmrE molecules in the dimer have different topologies, i.e. monomers have opposite orientations with respect to the membrane plane. A 3-D structure of EmrE acquired by electron cryo-microscopy (cryo-EM) at 7.5 Angstroms resolution in the membrane plane showed that parts of the structure are related by quasi-symmetry. We used this symmetry relationship, combined with sequence conservation data, to assign the transmembrane segments in EmrE to the densities seen in the cryo-EM structure. A C alpha model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix. The model is validated by much of the biochemical data on EmrE with most of the positions that were identified as affecting substrate translocation being located around the substrate-binding cavity. A suggested mechanism for proton-coupled substrate translocation in small multidrug resistance antiporters provides a mechanistic rationale to the experimentally observed inverted topology.
14646543
Parallel topology of genetically fused EmrE homodimers
EmrE is a small H+‐coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent‐solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C‐terminus of the first to N‐terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton‐dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.
13647122
MAS solid-state NMR studies on the multidrug transporter EmrE.
We study the uniformly 13C,15N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.
205299678
Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters*
Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N- and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.
40603302
Structural and functional aspects of the multidrug efflux pump AcrB
Abstract The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediates.
205849909
Drug transport mechanism of the AcrB efflux pump.
In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude cytotoxic substances from the cell directly into the medium bypassing periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump that extrudes multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB, a member of the Resistance Nodulation cell Division (RND) family, is the major site for substrate recognition and energy transduction of the entire tripartite system. The drug/proton antiport processes in this secondary transporter are suggested to be spatially separated, a feature frequently observed for primary transporters like membrane-bound ATPases. The recently elucidated asymmetric structure of the AcrB trimer reveals three different monomer conformations proposed to represent consecutive states in a directional transport cycle. Each monomer shows a distinct tunnel system with entrances located at the boundary of the outer leaflet of the inner membrane and the periplasm through the periplasmic porter (pore) domain towards the funnel of the trimer and TolC. In one monomer a hydrophobic pocket is present which has been shown to bind the AcrB substrates minocyclin and doxorubicin. The energy conversion from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (D407/D408/K940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel drug transport model combines the alternate access pump mechanism with the rotating site catalysis of F(1)F(o) ATPase as originally postulated by Jardetzky and Boyer, respectively, and suggests a working hypothesis for the transport mechanism of RND transporters in general.
26956908
Multidrug efflux transporter, AcrB--the pumping mechanism.
Resistance nodulation cell division (RND) transporters are one of the main causes of the bacterial multidrug resistance. They pump a wide range of antibiotics out of the cell by proton motive force. AcrB is the major RND transporter in Escherichia coli. Recently, the crystal structures of AcrB have been determined by different space groups. All these structures are consistent with asymmetric trimer. Each monomer has different conformation corresponding to one of the three functional states of the transport cycle. Transporting hydrophobic drug was bound in the periplasmic domain on one of the three monomers. The transport pathway with alternating access mechanism is located at the hydrophilic domain protruded into the periplasmic space while this mechanism of other transporter families like ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporter is located in the membrane-embedded region. For the RND, protonation might also take place asymmetrically at the functionally important charged residues in the transmembrane (TM) region. The structures indicate that drugs are transported by a three-step functional rotation in which substrates undergo ordered binding change.
2700884
Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism
The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.
2350621
Crystal structures of a multidrug transporter reveal a functionally rotating mechanism
AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB–drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.
4429773
Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels
ClC Cl- channels make up a large molecular family, ubiquitous with respect to both organisms and cell types. In eukaryotes, these channels fulfill numerous biological roles requiring gated anion conductance, from regulating skeletal muscle excitability to facilitating endosomal acidification by (H+)ATPases. In prokaryotes, ClC functions are unknown except in Escherichia coli, where the ClC-ec1 protein promotes H+ extrusion activated in the extreme acid-resistance response common to enteric bacteria. Recently, the high-resolution structure of ClC-ec1 was solved by X-ray crystallography. This primal prokaryotic ClC structure has productively guided understanding of gating and anion permeation in the extensively studied eukaryotic ClC channels. We now show that this bacterial homologue is not an ion channel, but rather a H+-Cl- exchange transporter. As the same molecular architecture can support two fundamentally different transport mechanisms, it seems that the structural boundary separating channels and transporters is not as clear cut as generally thought.
4396206
X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity
The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 Å, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with α-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.
41100297
A provisional transport mechanism for a chloride channel-type Cl−/H+ exchanger
Chloride channel (CLC)-type Cl−/H+ exchangers are widespread throughout the biological world, and one of these, CLC-ec1 from Escherichia coli, has been extensively studied. The structure of this protein is known, and several of its mechanistic hot spots have been identified, but a mechanism for Cl−/H+ exchange has not previously been offered. We herein confirm by direct measurements of Cl− and H+ fluxes a Cl−-to-H+ exchange stoichiometry of 2, and summarize experimental facts pertinent to the exchange mechanism. While the mechanism must involve a conformational cycle of alternating exposure of substrate-binding sites to the two sides of the membrane, CLC transporters do not adhere to a familiar ping-pong scheme in which the two ions bind in a mutually exclusive fashion. Instead, Cl− and H+ occupy the ion-binding region simultaneously. A conformational cycle is proposed that accounts for the exchange stoichiometry, several key mutants and the tendency of the protein to become uncoupled and allow ‘slippage’ of Cl−.
205241085
Ion permeation through a Cl−-selective channel designed from a CLC Cl−/H+ exchanger
The CLC family of Cl−-transporting proteins includes both Cl− channels and Cl−/H+ exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl− ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu148 and Tyr445, are known to impair H+ movement while preserving Cl− transport. In the x-ray crystal structure of CLC-ec1, these residues form putative “gates” flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H+ transport is abolished, and unitary Cl− transport rates are greatly increased, well above values expected for transporter mechanisms. Cl− transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl− flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.
6650297
Electrophoretic control of reconstituted adenine nucleotide translocation.
The initial velocity of adenine nucleotide exchange catalyzed by the reconstituted ADP-ATP carrier from beef heart mitochondria was measured under the influence of membrane potential and with different nucleotide distributions between the internal liposomal and the external buffer volume. Both Vmax and Km of adenine nucleotide uptake not only changed due to the applied potential but also depended on the respective nucleotide distribution. The rate equations for the ADP-ATP exchange under the various conditions were derived. These equations were simplified by assuming two alternative situations; either (a) af affinity type model, where the membrane potential influences only the affinity of the adenine nucleotide carrier toward ATP and ADP, or (b) a velocity type or distribution model, where the membrane potential modulates the rate constants of the ADP-ATP exchange. On the basis of several simplifications in the reconstituted system, the rate equations could be solved and the rate constants and dissociation constants of the exchange in the "energized" and in the "deenergized" state could be calculated. These values were used to derive prediction tables for normalized exchange rates under different nucleotide distributions, which were then compared with the experimental data. Only the exchange rates predicted by the velocity-type model agreed with the measured values. On the basis of this model a definite asymmetry caused by the membrane potential could be seen. Whereas this asymmetry is not very pronounced in the case of carrier-ADP complexes, about 40 times more ATP-loaded binding sites face the outside of the vesicles in the energized state.
41625033
Precursor/product antiport in bacteria
Many microorganisms metabolize their substrates (precursors) only partially and excrete the products of the metabolism into the medium. Although uptake of precursor and exit of product can proceed as two independent steps, there is increasing evidence that these processes are often linked and that transport is facilitated by a single antiport mechanism. Features of antiport mechanisms and advantages for the organism of catalysing precursor/product antiport will be illustrated by discussing a number of well‐characterized systems. Based on precursor‐product conversion stoichiometries, structural relatedness between precursors and products, and energetic and kinetic considerations, new examples of antiport systems will be proposed.
21901021
Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies
SummaryThe cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus laevis oocytes. Presteady-state and steady-state currents associated with cotransporter activity were measured with the two-electrode voltage-clamp technique. Steady-state sugar-dependent currents were measured between −150 and +90 mV as a function of external Na+ ([Na]0) and α-methyl-d-glucopyranoside concentrations ([αMDG]0). K0.5αMDGwas found to be dependent upon [Na]0 and the membrane potential. At Vm=−50 mV, increasing [Na]0 from 10 to 100 mm decreased K0.5αMDGfrom 1.5 mm to 180 μm. Increasing membrane potential toward negative values decreased K0.5αMDGat nonsaturating [Na]0. For instance, at 10 mM [Na]0, K0.5αMDGdecreased from 1.5 mm to 360 μm on increasing the membrane potential from −50 to −150 mV. The imaxαMDGwas relatively insensitive to [Na]0 between 10 and 100 mm and weakly voltage dependent (e-fold increase per 140 mV). K0.5Naand imaxNawere found to be dependent upon membrane potential and [sugar]0. In the presence of 1 mm [αMDG]0, K0.5Nadecreased from 50 to 5 mm between 0 and −150 mV and imaxNaincreased twofold between −30 and −200 mV. The voltage dependence of K0.5Nais consistent with an effect of potential on Na+ binding (Na+-well effect), whereas the voltage dependence of imaxNais compatible with the translocation step being voltage dependent. It is concluded that voltage influences both Na+ binding and translocation. Presteady-state currents were observed for depolarization pulses in the presence of 100 mm [Na]0. The transient current relaxed with a half time of =10 msec, and both the half time and magnitude of the transient varied with the holding potential and the size of depolarization pulse. Presteady-state currents were not observed after the addition of phlorizin or αMDG to the external Na+ solution and were not observed for water-injected control oocytes. We conclude that presteady-state currents are due to the activity of the carrier and that they may give a novel insight to the transport mechanism of the Na+/ glucose cotransporter.
9345004
Unraveling the mechanism of the lactose permease of Escherichia coli.
We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues-Glu-269, Arg-302, His-322, and Glu-325-that are the key participants in H(+) translocation and coupling between sugar and H(+) translocation. Although wild-type permease or mutants in Glu-325 and Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in either His-322 or Glu-269 do not titrate. The results offer a mechanistic model for lactose/H(+) symport. In the ground state, the permease is protonated, the H(+) is shared between His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and substrate is bound with high affinity at the outside surface. Substrate binding induces a conformational change that leads to transfer of the H(+) from His-322/Glu-269 to Glu-325 and reorientation of the binding site to the inner surface with a decrease in affinity. Glu-325 then is deprotonated on the inside because of rejuxtaposition with Arg-302. The His-322/Glu-269 complex then is reprotonated from the outside surface to reinitiate the cycle.
31325451
The kamikaze approach to membrane transport
Membrane transport proteins catalyse the movement of molecules into and out of cells and organelles, but their hydrophobic and metastable nature often makes them difficult to study by traditional means. Novel approaches that have been developed and applied to one membrane transport protein, the lactose permease from Escherichia coli, are now being used to study various other membrane proteins.
39652366
Towards an understanding of the structural basis of ‘forbidden’ transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport
Recent progress in the analysis of mutants of the Eschenchia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent ‘forbidden’ con‐formational changes. Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories. These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry. These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co‐substrate recognition and uncoupling are not independently variable.
21238052
An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport.
The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.
31752560
Uncoupling in Secondary Transport Proteins.
The kinetic behavior of a H-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (ESH) of the carrier also one of the binary complexes (ES or EH) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the EH leak type. The effects of pH and substrate concentration (pS) on the coupling of transport have been analyzed. In the enzyme with the ES leak, the proton:substrate stoichiometry (v(H)/v(S)) and the substrate accumulation levels decrease sigmoidally from fully coupled at low pH to completely uncoupled at high pH. Importantly, the coupling inferred from initial rate measurements is higher than from steady state accumulation levels. In the enzyme with the EH leak, the coupling inferred from the accumulation levels increases from no coupling at low pH to full coupling at high pH and saturating substrate concentration. The v(H)/v(S) increases sigmoidally with pH from <1 to >1 and is highly dependent on pS. At each pH value a substrate concentration can be found that results in apparent complete coupling between the two fluxes. The ES leak and the EH leak mutants provide a mechanism for substrate-induced and substrate-inhibited proton leakage, respectively. Furthermore, substrate efflux down a concentration gradient is inhibited by a membrane potential (inside negative) under uncoupled conditions in the case of an ES leak but not in the case of an EH leak. The properties of the mutants mimic those of various transport mutants that have been described, in particular mutants of the lactose transport protein of Escherichia coli. The analysis offers general means for targeted experimentation, which allows discrimination between various types of transport mutants.
3259383
Control of H+/Lactose Coupling by Ionic Interactions in the Lactose Permease ofEscherichia coli
A combinatorial approach was used to study putative interactions among six ionizable residues (Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325) in the lactose permease. Neutral mutations were made involving five ion pairs that had not been previously studied. Double mutants, R302L/E325Q and D240N/H322Q, had moderate levels of downhill [14C]-lactose transport. Mutants in which only one of these six residues was left unchanged (pentuple mutants) were also made. A Pent269− mutant (in which only Glu-269 remains) catalyzed a moderate level of downhill lactose transport. Pent240− and Pent 322+ also showed low levels of downhill lactose transport. Additionally, a Pent240− mutant exhibited proton transport upon addition of melibiose, but not lactose. This striking result demonstrates that neutralization of up to five residues of the lactose permease does not abolish proton transport. A mutant with neutral replacements at six ionic residues (hextuple mutant) had low levels of downhill lactose transport, but no uphill accumulation or proton transport. Since none of the mutants in this study catalyzes active accumulation of lactose, this is consistent with other reports that have shown that each residue is essential for proper coupling. Nevertheless, none of the six ionizable residues is individually required for substrate-induced proton cotransport. These results suggest that the H+ binding domain may be elsewhere in the permease or that cation binding may involve a flexible network of charged residues.
786406
Delineating Electrogenic Reactions during Lactose/H+ Symport†
Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H+ binding site (this step with a rate constant of ∼200 s−1 for wild-type LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s−1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical−biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.
16303860
Electrophysiological characterization of LacY
Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H+ symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H+ symport is H+ release. In contrast, LacY mutants Glu-325→Ala and Cys-154→Gly, which bind ligand normally, but are severely defective with respect to lactose/H+ symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H+ symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H+ during downhill sugar/H+ symport, which is the limiting step for this mode of transport.
27237900
Transport viewed as a catalytic process.
Transport catalysis is analysed in terms of the "induced transition fit" (ITF) concept. The essentials of ITF are briefly elucidated, emphasizing the difference of substrate-protein interactions between enzymes and carriers exemplified by the paradigm ADP/ATP carrier (AAC). Two of the numerous applications of the ITF are discussed in more detail: unidirectional passive and active transport and the relation of substrate site type inhibitors to the carrier conformations. According to ITF in most cases of unidirectional transport intrinsic binding energies may be insufficient for transport catalysis and requires additional energy from ATP or electrochemical gradients. The impacts of external energy on the carrier cycle are examined for ABC transporters (mdr) and for cation-substrate co-transporters (LacY). The relations of inhibitors to the binding site of the carrier are discussed, given the paradigm examples of side specific inhibitors of the AAC. Results with the AAC suggest the induction of an abortive ground state by inhibitors, representing extreme side specific conformation of the binding center.
84144144
Identification of a Lithium Interaction Site in the γ-Aminobutyric Acid (GABA) Transporter GAT-1*
The sodium- and chloride-dependent electrogenic γ-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuTAa. In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354.Atvaryingextracellularsodium,lithiumstimulatedsodiumdependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.
43310835
Critical Amino Acid Residues in Transmembrane Span 7 of the Serotonin Transporter Identified by Random Mutagenesis*
Transmembrane span 7 of the rat brain serotonin transporter was subjected to random mutagenesis. Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonconservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; Gly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitutions were preferred. Mutations that did not preserve these structural characteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side of the putative α-helix, lending support to this structural prediction. Mutations at some of these positions also specifically impaired transport activity under low Na+ conditions. Other mutations at nearby positions in transmembrane span 7 also impaired activity in low Na+, although the activity of the mutants in high Na+ was similar to wild type. These results suggest that at least some of the six critical residues play a role in Na+ binding or perhaps in the coupling of Na+ binding to later steps in the transport cycle. These residues may be important in other aspects of the transporter’s function as well.
6789169
Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1
To investigate the peculiar ionic specificity of KAAT1, an Na+- and K+-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na+-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K+ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na+, appeared to be completely abolished in the presence of K+. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the γ-aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K+, in addition to Na+, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.
12772409
Ion Binding and Permeation at the GABA Transporter GAT1
This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3H]GABA and [3H]tiagabine binding. Voltage jumps, Na+ and Cl− concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na+ ions interact with the transporter even in the absence of GABA, and (2) that Cl−facilitates the binding of Na+. Comparison between the charge movements and the transport-associated current shows that this initial Na+-transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation—treatment with high-affinity uptake blockers and the W68L mutation—“lock” Na+ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na+ substitutes Li+and Cs+ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na+ and Cl−, and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.
28446796
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.
21861764
Molecular mechanism of ion-ion and ion-substrate coupling in the Na+-dependent leucine transporter LeuT.
Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na(+) and not to Li(+) or K(+) cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na(+) in the case of double ion occupancy.
18177906
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 mus 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.
25230951
Amino Acid Residues in Transmembrane Segment IX of the Na+/I– Symporter Play a Role in Its Na+ Dependence and Are Critical for Transport Activity*
The Na+/I– symporter (NIS) is a key plasma membrane glycoprotein that mediates Na+-dependent active I– transport in the thyroid, lactating breast, and other tissues. The OH group of the side chain at position 354 in transmembrane segment (TMS) IX of NIS has been demonstrated to be essential for NIS function, as revealed by the study of the congenital I– transport defect-causing T354P NIS mutation. TMS IX has the most β-OH group-containing amino acids (Ser and Thr) of any TMS in NIS. We have thoroughly characterized the functional significance of all Ser and Thr in TMS IX in NIS, as well as of other residues in TMS IX that are highly conserved in other transporters of the SLC5A protein family. Here we show that five β-OH group-containing residues (Thr-351, Ser-353, Thr-354, Ser-356, and Thr-357) and Asn-360, all of which putatively face the same side of the helix in TMS IX, plus Asp-369, located in the membrane/cytosol interface, play key roles in NIS function and seem to be involved in Na+ binding/translocation.
31936377
The Aqueous Accessibility in the External Half of Transmembrane Domain I of the GABA Transporter GAT-1 Is Modulated by Its Ligands*
The sodium- and chloride-dependent γ-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052–37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membrane-embedded α-helix.
43342828
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.
8491962
Identification of a chloride ion binding site in Na+/Cl−-dependent transporters
The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl− independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl− ions. However, the only Cl− ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pKAs and homology modeling to predict the location of a functionally important Cl− binding site in serotonin transporter and other Cl−-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl− ion and through the observation of sequence conservation patterns in other Cl−-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl− and Na+ ions during transport. Other implications of the model are also discussed.
4391735
Mechanism of chloride interaction with neurotransmitter:sodium symporters
Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (γ-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.
21545107
Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1.
The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.
36474575
The equivalent of a thallium binding residue from an archeal homolog controls cation interactions in brain glutamate transporters
Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na+ followed by countertransport of K+. Two Tl+ binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K+-loaded liposomes was impaired. However, with Na+ and unlabeled L-aspartate inside the liposomes, exchange levels were around 50–90% of those by wild-type. In further contrast to wild-type, where either substrate or K+ stimulated the anion conductance by the transporter, substrate but not K+ modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.
14743939
Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model
Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co- and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, GltPh, only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of GltPh as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in GltPh) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.
190309
Mechanism of Cation Binding to the Glutamate Transporter EAAC1 Probed with Mutation of the Conserved Amino Acid Residue Thr101*
The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na+ ions, one H+ ion, and one glutamate molecule into the cell, in exchange for one K+ ion. Na+ binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na+] dependence of this conductance as a read-out of Na+ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr101, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na+ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1WT, in the T101A mutant this [Na+] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na+ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na+ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr101 and Asp367. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.
10371665
Interactions of alkali cations with glutamate transporters
The transport of glutamate is coupled to the co-transport of three Na+ ions and the countertransport of one K+ ion. In addition to this carrier-type exchange behaviour, glutamate transporters also behave as chloride channels. The chloride channel activity is strongly influenced by the cations that are involved in coupled flux, making glutamate transporters representative of the ambiguous interface between carriers and channels. In this paper, we review the interaction of alkali cations with glutamate transporters in terms of these diverse functions. We also present a model derived from electrostatic mapping of the predicted cation-binding sites in the X-ray crystal structure of the Pyrococcus horikoshii transporter GltPh and in its human glutamate transporter homologue EAAT3. Two predicted Na+-binding sites were found to overlap precisely with the Tl+ densities observed in the aspartate-bound complex. A novel third site predicted to favourably bind Na+ (but not Tl+) is formed by interaction with the substrate and the occluding HP2 loop. A fourth predicted site in the apo state exhibits selectivity for K+ over both Na+ and Tl+. Notably, this K+ site partially overlaps the glutamate-binding site, and their binding is mutually exclusive. These results are consistent with kinetic and structural data and suggest a plausible mechanism for the flux coupling of glutamate with Na+ and K+ ions.
12592968
Identification of the third Na+ site and the sequence of extracellular binding events in the glutamate transporter.
The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na(+) ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt(ph). Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt(ph) (the apo state, as well as in the presence of Na(+), the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na(+) ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na(+) ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp(312), Thr(92), and Asn(310), along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na(+) ions, reveals a stable structure--showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na(+) ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.
20969682
The substrate specificity of a neuronal glutamate transporter is determined by the nature of the coupling ion
Glutamate transporters are essential for terminating synaptic transmission. Glutamate is translocated together with three sodium ions. In the neuronal glutamate transporter EAAC1, lithium can replace sodium. To address the question of whether the coupling ion interacts with the ‘driven’ substrate during co‐transport, the kinetic parameters of transport of the three substrates, l‐glutamate and d‐ and l‐aspartate by EAAC‐1 in sodium‐ and lithium‐containing media were compared. The major effect of the substitution of sodium by lithium was on Km. In the presence of sodium, the values for Km and Imax of these substrates were similar. In the presence of lithium, the Km for l‐aspartate was increased around 13‐fold. Remarkably, the corresponding increase for l‐glutamate and d‐aspartate was much larger, around 130‐fold. In marked contrast, the Ki values for a non‐transportable substrate analogue were similar in the presence of either sodium or lithium. The preference for l‐aspartate in the presence of lithium was also observed when electrogenic transport of radioactive substrates was monitored in EAAC1‐containing proteoliposomes. Our results indicate that, subsequent to substrate binding, the co‐transported solutes interact functionally in the binding pocket of the transporter.
14336552
Macroscopic and Microscopic Properties of a Cloned Glutamate Transporter/Chloride Channel
The behavior of a Cl− channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of ∼5Å. Glutamate flux exhibited a much greater temperature dependence than Cl− flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl− conductance and a maximum channelPo ≪ 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of l-glutamate or d-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle.
7484642
Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier Eaac1
Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-μs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.
7615343
Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli.
Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H+ and/or sugar recognition. To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g. aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis. At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow. The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective. Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides. Mutations at position 325 had a markedly different phenotype. For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent Km for lactose transport (e.g. 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM). In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange. Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient. However, unlike the position 269 mutants, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-beta-D-galactopyranosyl-(1,1)-beta-thiogalactopyranoside, 1-O-methyl-beta-D-galactopyranoside, or melibiose. Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to delta pH. Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H+ transport via the lactose permease. Alternative hypotheses are also discussed.
43299600
Role of glutamate-269 in the lactose permease of Escherichia coli.
Glu-269, which is located on the hydrophilic face of putative helix VIII in the lactose permease of Escherichia coli, has been replaced with Asp, Gln or Cys by oligonucleotide-directed, site specific mutagenesis. Cells expressing Asp-269 permease exhibit no lactose accumulation or lactose-induced H+ translocation, but retain some ability to mediate lactose influx down a concentration gradient at high substrate concentrations. Furthermore, right-side-out membrane vesicles containing Asp-269 permease do not catalyse active lactose transport, facilitated lactose efflux or equilibrium exchange. Remarkably, however, Asp-269 permease accumulates beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside in a partially uncoupled fashion, whereas no transport of methyl-beta,D-thiogalactopyranoside, sucrose or maltose is detectable. Mutant permeases containing neutral replacements (Gln or Cys) or Glu-269 are completely devoid of activity, although the proteins are present in the membrane at concentrations comparable with wild-type or Asp-269 permease. The observations demonstrate that a carboxylate at position 269 is essential for transport activity, and Glu-269 is important for substrate binding and/or recognition.
34416776
Proton transport pathway in the ClC Cl-/H+ antiporter.
A fundamental question concerning the ClC Cl-/H+ antiporters is the nature of their proton transport (PT) pathway. We addressed this issue by using a novel computational methodology capable of describing the explicit PT dynamics in the ClC-ec1 protein. The main result is that the Glu203 residue delivers a proton from the intracellular solution to the core of ClC-ec1 via a rotation of its side chain and subsequent acid dissociation. After reorientation of the Glu203 side chain, a transient water-mediated PT pathway between Glu203 and Glu148 is established that is able to receive and translocate the proton via Grotthuss shuttling after deprotonation of Glu203. A molecular-dynamics simulation of an explicit hydrated excess proton in this pathway suggests that a negatively charged Glu148 and the central Cl- ion act together to drive H+ to the extracellular side of the membrane. This finding is consistent with the experimental result that Cl- binding to the central site facilitates the proton movement. A calculation of the PT free-energy barrier for the ClC-ec1 E203V mutant also supports the proposal that a dissociable residue is required at this position for efficient delivery of H+ to the protein interior, in agreement with recent experimental results.
28064
Neuronal and Glial Glycine Transporters Have Different Stoichiometries
A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs).