Structural dynamics of the monoamine transporter homolog LeuT from accelerated conformational sampling and channel analysis

The bacterial leucine transporter LeuT retains significant secondary structure similarities to the human monoamine transporters (MAT) such as the dopamine and serotonin reuptake proteins. The primary method of computational study of the MATs has been through the use of the crystallized LeuT structure. Different conformations of LeuT can give insight into mechanistic details of the MAT family. A conformational sampling performed through accelerated molecular dynamics simulations testing different combinations of the leucine substrate and bound sodium ions revealed seven distinct conformational clusters. Further analysis has been performed to target salt‐bridge residues R30–D404, Y108–F253, and R5–D369 and transmembrane domains on both the seven isolated structures and the total trajectories. In addition, solvent accessibility of LeuT and its substrate binding pockets has been analyzed using a program for calculating channel radii. Occupation of the Na2 site stabilizes the outward conformation and should bind to the open outward conformation before the leucine and Na1 sodium while two possible pathways were found to be available for intracellular transport. Proteins 2014; 82:2289–2302. © 2014 Wiley Periodicals, Inc.     

The effect of the intracellular sodium level on the activity of amino acid transport systems L and A in SV40 3T3 cells

The rate of transport of phenylalanine and leucine, pertinent amino acids of System L, has been measured in SV40 3T3 cells as a function of the presence of Na+ ions during the reloading phase that precedes the influx determination. The presence of Na+ ions during the reloading phase resulted in an increase of the subsequent substrate influx through System L. This effect was related to the intracellular Na+ level and was found to be independent by the presence of a chemical sodium gradient outside-inside during influx determination; furthermore, this effect could not be ascribed to a difference between control and Na+-treated cells in the internal levels of those amino acids that participate in the exchange phenomena of transport System L. The transport of phenylalanine appeared to have the ability to accept Li+ for Na+ substitution in the 'trans' position. The presence of Na+ ions in the 'trans' position was not required to optimize the transport of System A-reactive substrates, whose influxes are dependent on the presence of the cation in 'cis' position. Analysis of the relationship between influx and substrate concentration indicated that the Na+-dependent increase of substrate influx was associated with an enlarged capacity of the high-affinity component of transport System L.     

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.     

GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.

We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl-) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, Ein and E(out). In the Ein state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the Eout state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one gamma-aminobutyric acid (GABA) molecule can bind from the extracellular side. When Ein sites are empty, a transition to the Eout state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the Eout state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *Ein state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the Ein state. GABA-GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the Ein state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current-voltage relations. Experimental results that are simulated accurately include (a) all current-voltage relations, (b) all substrate dependencies described to date, (c) cis-cis and cis-trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre-steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA-GABA exchange, and (i) the existence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular chloride.     

Chloride-dependence of amino acid transport in rabbit ileum

Chloride-dependence of influx across the brush-border membrane of distal rabbit ileum was examined for beta-alanine, 2-methylaminoisobutyric acid (MeAIB), leucine, lysine, proline and D-glucose. Influx of leucine at 2 mM and of D-glucose at 0.5 mM was chloride-independent indicating that substitution of isethionate for chloride has no unspecific effect on sodium gradient driven transport processes. In contrast influx of beta-alanine and MeAIB was totally dependent on the presence of chloride ions. In the absence of chloride, proline transport was reduced to 20% of its control level. This remaining transport can be accounted for by the function of the carrier of alpha-amino-monocarboxylic acids. Transport of leucine at 0.1 mM was reduced by absence of chloride. This is in accordance with the observation of leucine transport by the beta-alanine carrier. The kinetics of chloride and sodium activation of transport of MeAIB were examined at 1 mM MeAIB. Chloride activation was characterized by a Hill coefficient of 1 and a K1/2 of 23.5 mM, and sodium activation by a Hill coefficient of 2 and a K1/2 of 51 mM. Thus cotransport of chloride with an imino acid would be compatible with the known rheogenic nature of this transport. This study adds the imino acid carrier and the beta-alanine carrier to the group of chloride-dependent, epithelial amino acid transport systems.     

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, Asp³¹², Thr⁹², and Asn³¹⁰, 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.     

Structural biology of solute carrier (SLC) membrane transport proteins

The human solute carriers (SLCs) comprise over 400 different transporters, organized into 65 families (http://slc.bioparadigms.org/) based on their sequence homology and transport function. SLCs are responsible for transporting extraordinarily diverse solutes across biological membranes, including inorganic ions, amino acids, lipids, sugars, neurotransmitters and drugs. Most of these membrane proteins function as coupled symporters (co-transporters) utilizing downhill ion (H⁺ or Na⁺) gradients as the driving force for the transport of substrate against its concentration gradient into cells. Other members work as antiporters (exchangers) that typically contain a single substrate-binding site with an alternating access mode of transport, while a few members exhibit channel-like properties. Dysfunction of SLCs is correlated with numerous human diseases and therefore they are potential therapeutic drug targets. In this review, we identified all of the SLC crystal structures that have been determined, most of which are from prokaryotic species. We further sorted all the SLC structures into four main groups with different protein folds and further discuss the well-characterized MFS (major facilitator superfamily) and LeuT (leucine transporter) folds. This review provides a systematic analysis of the structure, molecular basis of substrate recognition and mechanism of action in different SLC family members.     

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.     

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.     

Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 μs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na(+)-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.     

Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel

Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analogue tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations: closed (P450cam-C), intermediate (P450cam-I), and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of helix F, while both helices F and G move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well-defined states, while P450cam-O shows evidence of a somewhat broader distribution of conformations and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multistep closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well-defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multistep pathway may have significant implications for a full understanding of substrate specificity, kinetics, and coupling of substrate binding to P450 function.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Porter domain opening and closing motions in the multi-drug efflux transporter AcrB

Acriflavine resistance protein B acts as the active transporter in the multi-drug efflux pump Acriflavine resistance proteins A / B - Tolerance to colicins protein in Escherichia coli. Within the same reaction cycle intermediate all Acriflavine resistance protein B X-ray structures display highly similar conformations of the substrate-recruiting and transporting porter domain. To assess if this structural homogeneity is an intrinsic feature of Acriflavine resistance protein B or stems from other causes we performed a series of six independent, unbiased 100 ns molecular dynamics simulations of membrane-embedded, asymmetric, substrate-free wild type Acriflavine resistance protein B in a 150 mM NaCl solution. We find the porter domain more flexible than previously assumed displaying clear opening and closing motions of the proximal binding pocket (L and T-state) and the exit of the drug transport channels (O-intermediate). Concurrently the hydrophobic binding pocket favors a closed conformation in all three protomers. Our findings suggest that the conformational homogeneity seen in the crystal structures is likely an effect of bound but structurally unresolved substrate. Our simulations further imply that each of the known three reaction cycle intermediates occurs in at least two variants, the Thr676 loop independently regulates porter domain access and likely plays a key role in substrate transport. On a 100 ns time scale we find no evidence supporting the proposed LLL resting state in the absence of substrate. If the proximal binding pocket dynamics have an inhibiting effect on Acriflavine resistance protein B pump activity lowering the life time of substrate-accessible conformations, the observed dynamics could provide a structural explanation for the Acriflavine resistance protein B activity-enhancing effect of the adaptor protein Acriflavine resistance protein A stabilizing PC1 and PC2 subdomain orientations.     

Mechanistic studies of the apical sodium‐dependent bile acid transporter

In mammals, the apical sodium‐dependent bile acid transporter (ASBT) is responsible for the reuptake of bile acid from the intestine, thus recycling bile acid that is secreted from the gallbladder, for the purpose of digestion. As bile acid is synthesized from cholesterol, ASBT inhibition could have important implications in regulation of cholesterol levels in the blood. We report on a simulation study of the recently resolved structures of the inward‐facing ASBT from Neisseria meningitidis and from Yersinia frederiksenii, as well as of an ASBT variant from Yersinia frederiksenii suggested to be in the outward‐facing conformation. Classical and steered atomistic simulations and comprehensive potential of mean force analyses of ASBT, both in the absence and presence of ions and substrate, allow us to characterize and gain structural insights into the Na⁺ binding sites and propose a mechanistic model for the transport cycle. In particular, we investigate structural features of the ion translocation pathway, and suggest a third putative Na⁺ binding site. Our study sheds light on the structure–function relationship of bacterial ASBT and may promote a deeper understanding of transport mechanism altogether. Proteins 2015; 83:1107–1117. © 2015 Wiley Periodicals, Inc.     

Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria

We have determined and refined the X-ray crystal structures of six periplasmic binding proteins that serve as initial receptors for the osmotic-shock sensitive, active transport of L-arabinose, D-galactose/D-glucose, maltose, sulphate, leucine/isoleucine/valine and leucine. The tertiary structures and atomic interactions between proteins and ligands show common features that are important for understanding the function of the binding proteins. All six structures are ellipsoidal, consisting of two similar, globular domains. The ligand-binding site is located deep in the cleft between the two domains. Irrespective of the nature of the ligand (e.g. saccharide, sulphate dianion or leucine zwitterion), the specificities and affinities of the binding sites are achieved mainly through hydrogen-bonding interactions. Binding of ligands induces a large protein conformational change. Three different structures have been observed among the binding proteins: unliganded 'open cleft', liganded 'open cleft', and liganded 'closed cleft'. Here we discuss the functions of binding proteins in the light of numerous crystallographic and ligand-binding studies and propose a mechanism for the binding protein-dependent, high-affinity active transport.     

Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

The role of local hydration and hydrogen-bonding dynamics in ion and solute release from ion-coupled secondary transporters

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.     

Molecular mechanism of ligand recognition by membrane transport protein,Mhp1

The hydantoin transporter Mhp1 is a sodium‐coupled secondary active transport protein of the nucleobase‐cation‐symport family and a member of the widespread 5‐helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site‐directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5‐substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5‐substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5‐(2‐naphthylmethyl)‐L‐hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.     

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda)

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.     

Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway

We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.