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Kaspar P Locher - One of the best experts on this subject based on the ideXlab platform.
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structural basis of trans inhibition in a molybdate tungstate ABC Transporter
Science, 2008Co-Authors: Sabina Gerber, Mireia Comellasbigler, Birke A Goetz, Kaspar P LocherAbstract:Transport across cellular membranes is an essential process that is catalyzed by diverse membrane transport proteins. The turnover rates of certain Transporters are inhibited by their substrates in a process termed trans-inhibition, whose structural basis is poorly understood. We present the crystal structure of a molybdate/tungstate ABC Transporter (ModBC) from Methanosarcina acetivorans in a trans-inhibited state. The regulatory domains of the nucleotide-binding subunits are in close contact and provide two oxyanion binding pockets at the shared interface. By specifically binding to these pockets, molybdate or tungstate prevent adenosine triphosphatase activity and lock the Transporter in an inward-facing conformation, with the catalytic motifs of the nucleotide-binding domains separated. This allosteric effect prevents the Transporter from switching between the inward-facing and the outward-facing states, thus interfering with the alternating access and release mechanism.
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asymmetry in the structure of the ABC Transporter binding protein complex btucd btuf
Science, 2007Co-Authors: Rikki N Hvorup, Birke A Goetz, Martina Niederer, Kaspar Hollenstein, Eduardo Perozo, Kaspar P LocherAbstract:BtuCD is an adenosine triphosphate–binding cassette (ABC) Transporter that translocates vitamin B 12 from the periplasmic binding protein BtuF into the cytoplasm of Escherichia coli. The 2.6 angstrom crystal structure of a complex BtuCD-F reveals substantial conformational changes as compared with the previously reported structures of BtuCD and BtuF. The lobes of BtuF are spread apart, and B 12 is displaced from the binding pocket. The transmembrane BtuC subunits reveal two distinct conformations, and the translocation pathway is closed to both sides of the membrane. Electron paramagnetic resonance spectra of spin-labeled cysteine mutants reconstituted in proteoliposomes are consistent with the conformation of BtuCD-F that was observed in the crystal structure. A comparison with BtuCD and the homologous HI1470/71 protein suggests that the structure of BtuCD-F may reflect a posttranslocation intermediate.
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structure of an ABC Transporter in complex with its binding protein
Nature, 2007Co-Authors: Kaspar Hollenstein, Dominik C Frei, Kaspar P LocherAbstract:The structure of a putative molybdate Transporter (ModB2C2) is presented in complex with its cognate binding protein ModA. These results help provide the structural basis for the ATP-driven transport mechanism of both clinically relevant multi-drug ABC exporters and of ABC importers facilitating bacterial nutrient uptake. ATP-binding cassette (ABC) Transporter proteins carry diverse substrates across cell membranes1. Whereas clinically relevant ABC exporters are implicated in various diseases or cause multidrug resistance of cancer cells2,3, bacterial ABC importers are essential for the uptake of nutrients4, including rare elements such as molybdenum. A detailed understanding of their mechanisms requires direct visualization at high resolution and in distinct conformations. Our recent structure of the multidrug ABC exporter Sav1866 has revealed an outward-facing conformation of the transmembrane domains coupled to a closed conformation of the nucleotide-binding domains, reflecting the ATP-bound state5. Here we present the 3.1 A crystal structure of a putative molybdate Transporter (ModB2C2) from Archaeoglobus fulgidus in complex with its binding protein (ModA). Twelve transmembrane helices of the ModB subunits provide an inward-facing conformation, with a closed gate near the external membrane boundary. The ATP-hydrolysing ModC subunits reveal a nucleotide-free, open conformation, whereas the attached binding protein aligns the substrate-binding cleft with the entrance to the presumed translocation pathway. Structural comparison of ModB2C2A with Sav1866 suggests a common alternating access and release mechanism, with binding of ATP promoting an outward-facing conformation and dissociation of the hydrolysis products promoting an inward-facing conformation.
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structure of the multidrug ABC Transporter sav1866 from staphylococcus aureus in complex with amp pnp
FEBS Letters, 2007Co-Authors: Roger J P Dawson, Kaspar P LocherAbstract:Staphylococcus aureus Sav1866 is a bacterial homolog of the human ABC Transporter Mdr1 that causes multidrug resistance in cancer cells. We report the crystal structure of Sav1866 in complex with adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP) at 3.4 A resolution and compare it with the previously determined structure of Sav1866 with bound ADP. Besides differences in the ATP-binding sites, no significant conformational changes were observed. The results confirm that the ATP-bound state of multidrug ABC Transporters is coupled to an outward-facing conformation of the transmembrane domains.
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an inward facing conformation of a putative metal chelate type ABC Transporter
Science, 2007Co-Authors: Heather W Pinkett, Kaspar P Locher, Douglas C ReesAbstract:The crystal structure of a putative metal-chelate–type adenosine triphosphate (ATP)–binding cassette (ABC) Transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC Transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.
Chris Whitfield - One of the best experts on this subject based on the ideXlab platform.
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periplasmic depolymerase provides insight into ABC Transporter dependent secretion of bacterial capsular polysaccharides
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sean D Liston, S A Mcmahon, Michael D Suits, James H Naismith, Chris WhitfieldAbstract:Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces “Vi antigen” CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) Transporter. These Transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the Transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales , which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-A crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel β-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC Transporter-dependent export model. When introduced into S . Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC Transporter-dependent systems is actually exposed to the periplasm during envelope translocation.
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architecture of a channel forming o antigen polysaccharide ABC Transporter
Nature, 2018Co-Authors: Yunchen Bi, Chris Whitfield, Evan Mann, Jochen ZimmerAbstract:The crystal structure of a channel-forming O-antigen polysaccharide ABC Transporter suggests a novel biopolymer translocation mechanism. Bacterial cells are decorated with polysaccharides such as O-antigens, which help them to evade the innate immune responses of the host. In Gram-negative bacteria, these polysaccharides are transported from the cytoplasm to the periplasm before being incorporated into the outer membrane. In this paper, Jochen Zimmer and colleagues report the crystal structure of a bacterial O-antigen polysaccharide Transporter. This represents a key structure in bacterial cell envelope biogenesis. Unusually for ATP-binding cassette (ABC) Transporters, which usually operate by an alternating access model, the O-antigen Transporter in its open state forms a continuous channel which spans the entire membrane. As a result, the authors suggest that the polysaccharides are transported via a processive mechanism whereby they thread through the open channel. O-antigens are cell surface polysaccharides of many Gram-negative pathogens that aid in escaping innate immune responses1. A widespread O-antigen biosynthesis mechanism involves the synthesis of the lipid-anchored polymer on the cytosolic face of the inner membrane, followed by transport to the periplasmic side where it is ligated to the lipid A core to complete a lipopolysaccharide molecule2. In this pathway, transport to the periplasm is mediated by an ATP-binding cassette (ABC) Transporter, called Wzm–Wzt. Here we present the crystal structure of the Wzm–Wzt homologue from Aquifex aeolicus in an open conformation. The Transporter forms a transmembrane channel that is sufficiently wide to accommodate a linear polysaccharide. Its nucleotide-binding domain and a periplasmic extension form ‘gate helices’ at the cytosolic and periplasmic membrane interfaces that probably serve as substrate entry and exit points. Site-directed mutagenesis of the gates impairs in vivo O-antigen secretion in the Escherichia coli prototype. Combined with a closed structure of the isolated nucleotide-binding domains, our structural and functional analyses suggest a processive O-antigen translocation mechanism, which stands in contrast to the classical alternating access mechanism of ABC Transporters.
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structure biosynthesis and function of bacterial capsular polysaccharides synthesized by ABC Transporter dependent pathways
Carbohydrate Research, 2013Co-Authors: Lisa M Willis, Chris WhitfieldAbstract:Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) Transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC Transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.
Enrico Martinoia - One of the best experts on this subject based on the ideXlab platform.
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a rice ABC Transporter osABCc1 reduces arsenic accumulation in the grain
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Won Yong Song, Enrico Martinoia, Naoki Yamaji, Tomohiro Yamaki, Donghwi Ko, Kihong Jung, Miho Fujiikashino, Gynheung AnAbstract:Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) Transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.
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pdr type ABC Transporter mediates cellular uptake of the phytohormone abscisic acid
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Joohyun Kang, Jaeung Hwang, Sarah M Assmann, Enrico MartinoiaAbstract:Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA Transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) Transporter Arabidopsis thaliana Pleiotropic drug resistance Transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake Transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atABCg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atABCg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atABCg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants.
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an ABC Transporter mutation alters root exudation of phytochemicals that provoke an overhaul of natural soil microbiota
Plant Physiology, 2009Co-Authors: Dayakar V Badri, Enrico Martinoia, Naira Quintana, Elie El G Kassis, Young Hae Choi, Akifumi Sugiyama, Robert Verpoorte, Daniel K Manter, Jorge M VivancoAbstract:Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) Transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC Transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis ABCg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in ABCg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of ABCg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar Transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and ABCg30 cultured soils determined by pyrosequencing revealed that exudates from ABCg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.
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the ABC Transporter atABCb14 is a malate importer and modulates stomatal response to co2
Nature Cell Biology, 2008Co-Authors: Yongwook Choi, Enrico Martinoia, Bo Burla, Byeongwook Jeon, Masayoshi MaeshimaAbstract:Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC Transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.
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the ABC Transporter atpdr8 is a cadmium extrusion pump conferring heavy metal resistance
Plant Journal, 2007Co-Authors: Lucien Bovet, Enrico Martinoia, Masayoshi MaeshimaAbstract:Summary Cadmium (Cd) and lead (Pb) are widespread pollutants that are toxic to plant growth. The expression of AtPDR8 was upregulated in cadmium- or lead-treated Arabidopsis thaliana. To test whether AtPDR8 is involved in heavy metal resistance, we examined transgenic Arabidopsis that over-expressed AtPDR8 and RNAi plants that exhibited a severely reduced AtPDR8 transcript level, as well as T-DNA insertion mutants of this ABC Transporter. AtPDR8-over-expressing plants were more resistant to Cd2+ or Pb2+ than the wild-type and had lower Cd contents. In contrast, AtPDR8 RNAi transgenic plants and T-DNA insertion lines were more sensitive to Cd2+ or Pb2+ compared to wild-type plants and had higher Cd contents. The GFP–AtPDR8 protein was targeted to the plasma membrane, and GUS activity was present in most cells but strongest in the root hair and epidermal cells. Cd extrusion was higher in the AtPDR8-over-expressing plants in a flux assay using isolated protoplasts and radioactive 109Cd, and was lower in the RNAi transgenic plants than in the wild-type. Together, these data strongly support a role for AtPDR8 as an efflux pump of Cd2+ or Cd conjugates at the plasma membrane of Arabidopsis cells. As AtPDR8 has been suggested to be involved in the pathogen response and in the transport of chemicals that mediate pathogen resistance, this ABC protein is likely to transport a very broad range of substrates.
Jeanmichel Jault - One of the best experts on this subject based on the ideXlab platform.
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a multidrug ABC Transporter with a taste for gtp
Scientific Reports, 2018Co-Authors: Cedric Orelle, Claire Durmort, Khadija Mathieu, Benjamin Duchene, Sandrine Aros, Francois Fenaille, Francois Andre, Christophe Junot, Thierry Vernet, Jeanmichel JaultAbstract:During the evolution of cellular bioenergetics, many protein families have been fashioned to match the availability and replenishment in energy supply. Molecular motors and primary Transporters essentially need ATP to function while proteins involved in cell signaling or translation consume GTP. ATP-Binding Cassette (ABC) Transporters are one of the largest families of membrane proteins gathering several medically relevant members that are typically powered by ATP hydrolysis. Here, a Streptococcus pneumoniae ABC Transporter responsible for fluoroquinolones resistance in clinical settings, PatA/PatB, is shown to challenge this concept. It clearly favors GTP as the energy supply to expel drugs. This preference is correlated to its ability to hydrolyze GTP more efficiently than ATP, as found with PatA/PatB reconstituted in proteoliposomes or nanodiscs. Importantly, the ATP and GTP concentrations are similar in S. pneumoniae supporting the physiological relevance of GTP as the energy source of this bacterial Transporter.
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dynamics of a bacterial multidrug ABC Transporter in the inward and outward facing conformations
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Jeanmichel Jault, Shahid Mehmood, Carmen Domene, Eric ForestAbstract:The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) Transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related Transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.
Douglas C Rees - One of the best experts on this subject based on the ideXlab platform.
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noncanonical role for the binding protein in substrate uptake by the metni methionine atp binding cassette ABC Transporter
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Phong T Nguyen, Douglas C Rees, Jens T KaiserAbstract:The Escherichia coli methionine ABC Transporter MetNI exhibits both high-affinity transport toward l-methionine and broad specificity toward methionine derivatives, including d-methionine. In this work, we characterize the transport of d-methionine derivatives by the MetNI Transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward d-selenomethionine. We determined the crystal structure at 2.95 A resolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the Transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the Transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC Transporters to transport both high- and low-affinity substrates present in a physiological concentration range.
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an inward facing conformation of a putative metal chelate type ABC Transporter
Science, 2007Co-Authors: Heather W Pinkett, Kaspar P Locher, Douglas C ReesAbstract:The crystal structure of a putative metal-chelate–type adenosine triphosphate (ATP)–binding cassette (ABC) Transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC Transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.
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the e coli btucd structure a framework for ABC Transporter architecture and mechanism
Science, 2002Co-Authors: Kaspar P Locher, Douglas C ReesAbstract:The ABC Transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC Transporter mediating vitamin B_(12) uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC Transporters.
