Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin

ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.     

The Mechanism of Action of Multidrug-Resistance-Linked P-Glycoprotein

P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.     

Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA

MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.     

Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake

The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.     

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP hydrolysis to the transport of various molecules across cellular membranes. Found in both prokaryotes and eukaryotes, a sub-group of these transporters are involved in the efflux of hydrophobic drugs and lipids, causing anti-microbial and chemotherapeutic multidrug resistance. In this review, we examine recent structural and functional analysis of the ABC transporter MsbA and implications on the mechanism of multidrug efflux.     

The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.     

Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-γ-S demonstrated that physicochemical properties of the bilayer modulated binding and ATPase activity. This is remarkable when considering that the catalytic unit is located ~50? from the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules.     

Structure of MsbA from Vibrio cholera: a multidrug resistance ABC transporter homolog in a closed conformation

The spread of multidrug resistance (MDR) is a world health crisis that presents a significant challenge to the treatment of cancer and infection. MDR can be caused by a group of ABC (MDR-ABC) transporters that move hydrophobic drug molecules and lipids across the cell membrane. To gain insight into the conformational changes these transporters undergo when flipping hydrophobic substrates across the lipid bilayer, we have determined the structure of the lipid flippase MsbA from Vibrio cholera (VC-MsbA) to 3.8A. Structural comparison of VC-MsbA to MsbA from Escherichia coli reveals that the transporters share a structurally conserved core of transmembrane alpha-helices, but differ in the relative orientations of their nucleotide-binding domains (NBD). The transmembrane domain of VC-MsbA is captured in a closed conformation and the structure supports a "power stroke" model of transporter dynamics where opposing NBDs associate upon ATP binding. The separation of the alpha and beta domains of the NBD suggests the possibility that their association could make them competent to bind ATP and gives further insight into the structural basis for catalytic regulation.     

The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact,Head-to-Tail Configuration of the Nucleotide-Binding Domains

ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration ‘sandwich’ dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD ‘Switch’ mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis

We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes.     

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA

Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme–substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme–product complex (trapped by ADP-vanadate). 20 Å resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.     

Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA

ATP-binding cassette (ABC) transporters make up one of the largest superfamilies of proteins known and have been shown to transport substrates ranging from lipids and antibiotics to sugars and amino acids. The dysfunction of ABC transporters has been linked to human pathologies such as cystic fibrosis, hyperinsulinemia, and macular dystrophy. Several bacterial ABC transporters are also necessary for bacterial survival and transport of virulence factors in an infected host. MsbA is a 65 kDa protein that forms a functional homodimer consisting of two six-helix transmembrane domains and two approximately 250 amino acid nucleotide-binding domains (NBD). The NBDs contain several conserved regions such as the Walker A, LSGGQ, and H motif that bind directly to ATP and align it for hydrolysis. MsbA transports lipid A, its native substrate, across the inner membrane of Gram-negative bacteria. The loss or dysfunction of MsbA results in a toxic accumulation of lipid A inside the cell, leading to cell-membrane instability and cell death. Using site-directed spin labeling electron paramagnetic resonance spectroscopy, conserved motifs within the MsbA NBD have been evaluated for structure and dynamics upon substrate binding. It has been determined that the LSGGQ NBD consensus sequence is consistent with an alpha-helical conformation and that these residues maintain extensive tertiary contacts throughout hydrolysis. The dynamics of the LSGGQ and the H-motif region have been studied in the presence of ATP, ADP, and ATP plus vanadate to identify the residues that are directly affected by interactions with the substrate before, after, and during hydrolysis, respectively.     

Conformational Cycle of the ABC Transporter MsbA in Liposomes: Detailed Analysis Using Double Electron-Electron Resonance Spectroscopy

Driven by the energy of ATP binding and hydrolysis, ATP-binding cassette transporters alternate between inward- and outward-facing conformations, allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large-amplitude motion that disengages the nucleotide-binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and double electron-electron resonance spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20-30 Å closing motion while a 7- to 10-Å opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. Double electron-electron resonance distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate-binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work, providing a framework for the mechanism of substrate transport.     

Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP‐binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208′) in the intracellular domains of its two half‐transporters, close to the nucleotide‐binding domains (NBDs), which are in close proximity of each other in the outward‐facing state but not in the inward‐facing state. Using intermolecular cysteine crosslinking between E208C and E208C′ in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide‐free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half‐transporters like a nucleotide‐free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Evidence for a Molecular Diode-based Mechanism in a Multispecific ATP-binding cassette (ABC) Exporter: SER-1368 AS A GATEKEEPING RESIDUE IN THE YEAST MULTIDRUG TRANSPORTER Pdr5*

ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.