Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: Evidence for a carrier conformational change on complex formation

Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min^–1 and 0.33±0.02 min^–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Cryo-EM structure of a eukaryotic zinc transporter at a low pH suggests its Zn2+-releasing mechanism

Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H⁺-coupled uptake (antiport) of Zn²⁺ into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn²⁺. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn²⁺ at a luminal pH (5.5). Compared to a Zn²⁺-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn²⁺-binding site with a disrupted coordination geometry. Combined with a Zn²⁺-binding assay our data suggest that protons may disrupt Zn²⁺ coordination at the transmembrane Zn²⁺-binding site in the lumen-facing state, thus facilitating Zn²⁺ release from ZnT8 into the lumen.     

Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism

The human SLC28 family of concentrative (Na⁺-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNT_NW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter’s outward-facing conformation. In addition, both the Na⁺ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.     

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.     

Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn²⁺, but the nature of the Zn²⁺-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn²⁺ on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn²⁺ depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn²⁺ to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn²⁺ dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn²⁺ on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na⁺ concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn²⁺. This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).     

Asymmetric Conformational Flexibility in the ATP-Binding Cassette Transporter HI1470/1

Putative metal-chelate-type ABC transporter HI1470/1 is homologous with vitamin B₁₂ importer BtuCD but exhibits a distinct inward-facing conformation in contrast to the outward-facing conformation of BtuCD. Normal-mode analysis of HI1470/1 reveals the intrinsic asymmetric conformational flexibility in this transporter and demonstrates that the transition from the inward-facing to the outward-facing conformation is realized through the asymmetric motion of individual subunits of the transporter. This analysis suggests that the asymmetric arrangement of the BtuC dimer in the crystal structure of the BtuCD-F complex represents an intermediate state relating HI1470/1 and BtuCD. Furthermore, a twisting motion between transmembrane domains and nucleotide-binding domains encoded in the lowest-frequency normal mode of this type of importer is found to contribute to the conformational transitions during the whole cycle of substrate transportation. A more complete translocation mechanism of the BtuCD type importer is proposed.     

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

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

Nucleotide-free MalK Drives the Transition of the Maltose Transporter to the Inward-facing Conformation

The complex MalFGK₂ hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK₂ and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.     

Lactose permease and the alternating access mechanism

Crystal structures of the lactose permease of Escherichia coli (LacY) reveal 12, mostly irregular transmembrane α-helices surrounding a large cavity open to the cytoplasm and a tightly sealed periplasmic side (inward-facing conformation) with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm. However, LacY is highly dynamic, and binding of a galactopyranoside causes closing of the inward-facing cavity with opening of a complementary outward-facing cavity. Therefore, the coupled, electrogenic translocation of a sugar and a proton across the cytoplasmic membrane via LacY very likely involves a global conformational change that allows alternating access of sugar- and H(+)-binding sites to either side of the membrane. Here the various biochemical and biophysical approaches that provide strong support for the alternating access mechanism are reviewed. Evidence is also presented indicating that opening of the periplasmic cavity is probably the limiting step for binding and perhaps transport.     

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn²⁺ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn²⁺ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp⁵⁹⁹ and His⁴⁵¹, with alanine was sufficient to block Zn²⁺ transport. These findings indicate, for the first time, that Zn²⁺ transport mediated by a mammalian ZnT is catalyzed by H⁺/Zn²⁺ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.     

smFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA

Bacterial multidrug-resistance transporters of the major facilitator superfamily are distinguished by their extraordinary ability to bind structurally diverse substrates, thus serving as a highly efficient tool to protect cells from multiple toxic substances present in their environment, including antibiotic drugs. However, details of the dynamic conformational changes of the transport cycle involved remain to be elucidated. Here, we used the single-molecule fluorescence resonance energy transfer technique to investigate the conformational behavior of the Escherichia coli multidrug transporter MdfA under conditions of different substrates, pH, and alkali metal ions. Our data show that different substrates exhibit distinct effects on both the conformational distribution and transition rate between two major conformations. Although the cationic substrate tetraphenylphosphonium favors the outward-facing conformation, it has less effect on the transition rate. In contrast, binding of the electroneutral substrate chloramphenicol tends to stabilize the inward-facing conformation and decreases the transition rate. Therefore, our study supports the notion that the MdfA transporter uses distinct mechanisms to transport electroneutral and cationic substrates.     

Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1

We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Cryo-EM structure of AMP-PNP-bound human mitochondrial ATP-binding cassette transporter ABCB7

ATP-binding cassette subfamily B member 7 (ABCB7) is localized in the inner membrane of mitochondria, playing a critical role in iron metabolism. Here, we determined the structure of the nonhydrolyzable ATP analog adenosine-5′-(β-γ-imido) triphosphate (AMP-PNP) bound human ABCB7 at 3.3 Å by single-particle electron cryo-microscopy (cryo-EM). The AMP-PNP-bound human ABCB7 shows an inverted V-shaped homodimeric architecture with an inward-facing open conformation. One AMP-PNP molecule and Mg²⁺ were identified in each nucleotide-binding domain (NBD) of the hABCB7 monomer. Moreover, four disease-causing missense mutations of human ABCB7 have been mapped to the structure, creating a hotspot map for X-linked sideroblastic anemia and ataxia disease. Our results provide a structural basis for further understanding the transport mechanism of the mitochondrial ABC transporter.     

The conformational transition pathways of ATP-binding cassette transporter BtuCD revealed by targeted molecular dynamics simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B(12) into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the "alternating access" mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O→I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I→O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process.     

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP

The Na⁺-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na⁺-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.     

3D Cryo-Electron Reconstruction of BmrA,a Bacterial Multidrug ABC Transporter in an Inward-Facing Conformation and in a Lipidic Environment

ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.     

Structure and mechanism of ABC transporter proteins

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common 'coupling helix' that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters.