Outward-facing Conformation Research Articles

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

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 ASBTNM and ASBTYf 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 ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM 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 ASBTNM 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.

An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter.

Apical sodium-dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator-style alternating-access model, which was deduced mainly by comparing high-resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward-facing structure (PDB entry 4n7x) was obtained with an Na+-binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+- or substrate-binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild-type ASBTYf. These data support the hypothesis that the PDB entry 4n7x-like structure represents a functional outward-facing conformation of ASBTYf in its transport cycle.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3−, CO32−, and Cl− necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl−/HCO3− exchange versus Na+-CO32− symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein–ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32− binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Quercetin acts as a P-gp modulator via impeding signal transduction from nucleotide-binding domain to transmembrane domain

The inherent ability of the cancer cells to resist chemotherapeutic agents is a major challenge in drug discovery. Chemotherapy is one of the most widely used treatment methods for cancer, but due to multidrug resistance (MDR) development in cancer cells, the healing procedure often fails. Various factors impart cancer resistance to cells; among them, P-glycoprotein (P-gp) overexpression is directly linked to MDR in cancer cells. P-gp leads to the efflux of drug molecules to the extracellular space. Several molecules have been reported to inhibit the P-gp activity. Among them, quercetin has revealed a great potential to modulate P-gp activity. However, the mechanistic understanding of quercetin induced modulation is not entirely elucidated. In the present work, we showed that quercetin binds in the interacting region between the transmembrane domain and nucleotide-binding domain out of the three plausible binding sites of P-gp and restrict the conformational change from inward- to outward-facing conformation of P-gp. Due to the absence of the inward-facing structure of human P-gp, we first modeled an inward-facing P-gp structure. Using molecular docking, the interacting residues of P-gp were identified, and the stability and interaction dynamics of the complex were studied using molecular dynamics simulation. Our work reveals the mechanistic understanding of quercetin induced modulation of P-gp and indicates its importance in cancer treatment. Communicated by Ramaswamy H. Sarma

An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments.

Structural basis of ion transport and inhibition in ferroportin

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.

Capturing Substrate Translocation in an ABC Exporter at the Atomic Level.

ATP-binding cassette (ABC) transporters chemomechanically couple ATP binding and hydrolysis to large-scale conformational changes, ultimately leading to substrate translocation across biological membranes. Despite recent progress in the structure determination of substrate-bound ABC exporters, the inherently dynamic mechanism of substrate transport remains unclear at the atomic level. In this work, we capture substrate translocation in the heterodimeric ABC exporter TM287/288 from the hyperthermophilic bacterium Thermotoga maritima using all-atom molecular dynamics (MD) simulations. Unguided multimicrosecond simulations at 375 K show how the drugs daunorubicin and verapamil, which were initially docked into the ABC transporter, get translocated through the exporter by following its large-scale alternating access conformational transitions between an inward-facing (IF) and an outward-facing (OF) conformation. Triggered by the affinity difference due to differential solvation of the binding cavity in the IF and OF conformations, the substrates unbind from the OF transporter and partition into the lipid bilayer. While daunorubicin is stably inserted into the outer leaflet of the bilayer, verapamil dynamically flip flops between the bilayer leaflets, possibly rendering its net transport futile.

Ex vivo Quantitative Proteomic Analysis of Serotonin Transporter Interactome: Network Impact of the SERT Ala56 Coding Variant.

Altered serotonin (5-HT) signaling is associated with multiple brain disorders, including major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). The presynaptic, high-affinity 5-HT transporter (SERT) tightly regulates 5-HT clearance after release from serotonergic neurons in the brain and enteric nervous systems, among other sites. Accumulating evidence suggests that SERT is dynamically regulated in distinct activity states as a result of environmental and intracellular stimuli, with regulation perturbed by disease-associated coding variants. Our lab identified a rare, hypermorphic SERT coding substitution, Gly56Ala, in subjects with ASD, finding that the Ala56 variant stabilizes a high-affinity outward-facing conformation (SERT∗) that leads to elevated 5-HT uptake in vitro and in vivo. Hyperactive SERT Ala56 appears to preclude further activity enhancements by p38α mitogen-activated protein kinase (MAPK) and can be normalized by pharmacological p38α MAPK inhibition, consistent with SERT Ala56 mimicking, constitutively, a high-activity conformation entered into transiently by p38α MAPK activation. We hypothesize that changes in SERT-interacting proteins (SIPs) support the shift of SERT into the SERT∗ state which may be captured by comparing the composition of SERT Ala56 protein complexes with those of wildtype (WT) SERT, defining specific interactions through comparisons of protein complexes recovered using preparations from SERT–/– (knockout; KO) mice. Using quantitative proteomic-based approaches, we identify a total of 459 SIPs, that demonstrate both SERT specificity and sensitivity to the Gly56Ala substitution, with a striking bias being a loss of SIP interactions with SERT Ala56 compared to WT SERT. Among this group are previously validated SIPs, such as flotillin-1 (FLOT1) and protein phosphatase 2A (PP2A), whose functions are believed to contribute to SERT microdomain localization and regulation. Interestingly, our studies nominate a number of novel SIPs implicated in ASD, including fragile X mental retardation 1 protein (FMR1) and SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), of potential relevance to long-standing evidence of serotonergic contributions to ASD. Further investigation of these SIPs, and the broader networks they engage, may afford a greater understanding of ASD as well as other brain and peripheral disorders associated with perturbed 5-HT signaling.

The Role of Dopamine in the Stimulant Characteristics of Novel Psychoactive Substances (NPS)-Neurobiological and Computational Assessment Using the Case of Desoxypipradrol (2-DPMP).

Stimulant drugs, including novel psychoactive substances (NPS, formerly “legal highs”) have addictive potential which their users may not realize. Stimulants increase extracellular dopamine levels in the brain, including the reward and addiction pathways, through interacting with dopamine transporter (DAT). This work aimed to assess the molecular and atomistic mechanisms of stimulant NPS actions at DAT, which translate into biological outcomes such as dopamine release in the brain’s reward pathway. We applied combined in vitro, in vivo, and in silico methods and selected 2-diphenylmethylpiperidine (2-DPMP) as an example of stimulant NPS for this study. We measured in vitro binding of 2-DPMP to rat striatum and accumbens DAT by means of quantitative autoradiography with a selective DAT-radioligand [125I]RTI-121. We evaluated the effects of intravenously administered 2-DPMP on extracellular dopamine in the accumbens-shell and striatum using in vivo microdialysis in freely moving rats. We used dynamic modeling to investigate the interactions of 2-DPMP within DAT, in comparison with cocaine and amphetamine. 2-DPMP potently displaced the radioligand in the accumbens and striatum showing dose-dependence from 0.3 to 30 μM. IC50 values were: 5.65 × 10-7M for accumbens shell and 6.21 × 10-7M for dorsal striatum. Dose-dependent responses were also observed in accumbens-shell and striatum in vivo, with significant increases in extracellular dopamine levels. Molecular dynamics simulations identified contrasting conformational changes of DAT for inhibitors (cocaine) and releasers (amphetamine). 2-DPMP led to molecular rearrangements toward an outward-facing DAT conformation that suggested a cocaine-type effect. The present combination of molecular modeling with experimental neurobiological procedures allows for extensive characterization of the mechanisms of drug actions at DAT as the main molecular target of stimulants, and provides an insight into the role of dopamine in the molecular and neurobiological mechanisms of brain responses to stimulant NPS that have addictive potential. Such knowledge reveals the risk of addiction related to NPS use. The research presented here can be adapted for other psychostimulants that act at their membrane protein targets.

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux.

Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na+-coupled MFS-MDR transporter, MdrP, which also can achieve H+-coupled drug efflux independently of Na+. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na+ antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na+ and H+ coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na+ translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na+ and H+ coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.

Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses.

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.

Bile Acid Transport by the Symporter ASBTNM: Substrate Binding and Conformational Change

The apical sodium-dependent bile acid transporter (ASBT) allows reabsorption of bile acids from the intestine by coupling bile acid movement to the sodium gradient. Such transporters are attractive targets for drug delivery and in the treatment for hypercholesterolaemia. Several structures of bacterial homologues in both inward and outward facing conformations have been obtained experimentally, including a substrate-bound inward facing structure of the homologue from Neisseria meningitidis (ASBTNM). However, many details surrounding the binding of substrates and the conformational transition remain unclear. We have used experimental and computational methods to explore these details at an atomistic scale using the homologue ASBTNM and the bile acid taurocholate. Models of apo and substrate-bound ASBTNM in the outward-facing conformation were generated. Biased molecular dynamics simulations and free energy calculations were used to explore conformational changes and possible movement of taurocholate during the transition by an elevator mechanism. Probability distributions and the residues important for binding of sodium and taurocholate in wild type and mutant ASBTNM were investigated with unbiased simulations, revealing alternative sodium binding sites. These results further our understanding of the important molecular details of ASBT function.

High Speed AFM Imaging of Structure and Dynamics of Bacterial ABC Transporter MsbA During Lipid Transport

All ATP-binding cassette (ABC) transporters that operate in all species from bacteria to human share a common structural organization, consisting of two distinct domains (i.e., transmembrane domains (TMDs) and cytosolic nucleotide binding domains (NBDs)). MsbA, an ABC transporter, exports lipid A across the cytoplasmic membrane of Gram-negative bacteria. MsbA also exports drugs, conferring multidrug resistance. The crystal and cryo-EM snapshot structures of MsbA bound with different nucleotides have suggested that large conformational changes possibly take place in the TMDs accompanied the ATPase reaction in two NBDs. Therefore, the two NBDs must repeat coupling and uncoupling during multiple ATPase cycles. These dynamic events very likely transmit to the two α-helical bundles connected with the NBDs, so that the TMDs (major parts of the α-helical bundles) undergo different conformations including inward-facing and outward-facing conformations that facilitate substrate entry, translocation and export. However, this structure and dynamics of MsbA during these functional activities have not been verified simultaneously. Using HS-AFM advantage, the structures and dynamics of detergent-solubilized MsbA bound with different nucleotides were successfully real-time imaged to follow their conformational changes. The kinetics of open and closed conformations in NBDs and TMDs was analyzed to evaluate how coupling and uncoupling NBDs could change structural dynamics in TMDs. Next, structural dynamics of TMDs channel and NBDs of MsbA embedded within lipid membrane containing lipid A was also real-time imaged in the presence of different nucleotides and drugs. The translocation of lipid A across TMDs channel was successfully recorded in the controllably oriented MsbA molecules within lipid membrane. Taken together, the dynamic conformational changes in TMDs accompanying ATP hydrolysis in NBDs during lipid A transport unveil a very important operational mechanism in bacterial ABC transporters.

Simulations of Active Transport Across the Blood-Brain Barrier

The blood-brain barrier (BBB) is responsible for maintaining homeostasis of the brain by regulating the chemical environment, immune cell transport, and the entry of toxins and pathogens, and misregulation of the BBB has been shown to be implicated in numerous CNS disorders. One of the challenges in studying the BBB include the bioavailability of therapies, as well as rising multidrug resistance of pathogens. A major contributor to drug resistance and lacking bioavailability of therapies is the over-expression of the broad-specificity P-glycoprotein (P-gp) efflux pump in the endothelial cells of the BBB. However, despite the role of P-gp in affecting the bioavailability, pharmacokinetics and efficacy of therapeutic drugs, the mechanism of the efflux of substrates via P-gp, as well as the inhibition of P-gp activity, are not well understood. To address the mechanisms of P-gp-mediated substrate efflux and P-gp inhibition, we performed multimicrosecond unbiased atomic detail molecular dynamics (MD) simulations of the P-glycoprotein from novel human cryo-EM models as well as X-ray crystal structures in the presence of substrates (doxorubicin, rhodamine-123) and an inhibitor (tariquidar). We compare the differential transport gradient of P-gp substrates and inhibitors between the inward-facing (IF) and outward-facing (OF) conformations of P-gp to propose a model of P-gp turnover. We compare this model of active transport to our model of passive transport across the BBB.

Interplay Between Membrane Curvature and Conformational States in ABC Transporters

ATP-Binding Cassette (ABC) transporters constitute one of the largest superfamily of transmembrane proteins, present in both prokaryotes and eukaryotes. ATP hydrolysis at the nucleotide binding domains (NBD) drives conformational changes in the transmembrane domains (TMD) allowing substrate translocation. During this process, the structure of the ABC transporter alternates between the inward-facing (IF) conformation and the outward-facing (OF) conformation. The most prevalent and best characterized ABC-member is the mammalian P-glycoprotein (Pgp).

Modelling of Ion and Water Permeation in the Outward Facing Conformation of Human Anion Exchanger 1

Modelling of Ion and Water Permeation in the Outward Facing Conformation of Human Anion Exchanger 1