95 results on '"Klaas M. Pos"'
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2. Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms
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Alina Ornik-Cha, Julia Wilhelm, Jessica Kobylka, Hanno Sjuts, Attilio V. Vargiu, Giuliano Malloci, Julian Reitz, Anja Seybert, Achilleas S. Frangakis, and Klaas M. Pos
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Science - Abstract
Resistance-nodulation-cell division (RND)-type tripartite efflux pumps confer multidrug resistance to Gram-negative bacteria. Here, structural and functional analyses of AdeB from Acinetobacter baumannii and AcrB from Escherichia coli provide insight into their different drug-binding and conformational drug transport states.
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- 2021
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3. Allosteric drug transport mechanism of multidrug transporter AcrB
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Heng-Keat Tam, Wuen Ee Foong, Christine Oswald, Andrea Herrmann, Hui Zeng, and Klaas M. Pos
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Science - Abstract
Gram-negative bacteria can display intrinsic antibiotic resistance due to the action of tripartite efflux pumps, which include a H+/drug antiporter component. Here, the authors present a structure-function analysis of antiporter AcrB in intermediate states of the transport cycle, showing novel drug-binding sites and transport pathways.
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- 2021
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4. Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria
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Nina Compagne, Anais Vieira Da Cruz, Reinke T. Müller, Ruben C. Hartkoorn, Marion Flipo, and Klaas M. Pos
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efflux pump inhibitor ,RND multidrug efflux pump ,Gram-negative bacteria ,antimicrobial resistance ,antibiotic resistance breakers ,AcrB ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely resistant to known antibiotics. AMR is caused by several mechanisms, among which the (over)expression of multidrug efflux pumps plays a central role. Multidrug efflux pumps are transmembrane transporters, naturally expressed by Gram-negative bacteria, able to extrude and confer resistance to several classes of antibiotics. Targeting them would be an effective way to revive various options for treatment. Many efflux pump inhibitors (EPIs) have been described in the literature; however, none of them have entered clinical trials to date. This review presents eight families of EPIs active against Escherichia coli or Pseudomonas aeruginosa. Structure–activity relationships, chemical synthesis, in vitro and in vivo activities, and pharmacological properties are reported. Their binding sites and their mechanisms of action are also analyzed comparatively.
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- 2023
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5. Binding of Tetracyclines to Acinetobacter baumannii TetR Involves Two Arginines as Specificity Determinants
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Manuela Sumyk, Stephanie Himpich, Wuen Ee Foong, Andrea Herrmann, Klaas M. Pos, and Heng-Keat Tam
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transcription repressor ,antibiotic resistance ,Acinetobacter baumannii ,TetR family ,tetracycline transporter ,tetracycline ,Microbiology ,QR1-502 - Abstract
Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.
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- 2021
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6. Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium
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Jenifer Cuesta Bernal, Jasmin El-Delik, Stephan Göttig, and Klaas M. Pos
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antibiotic resistance ,efflux pump ,RND ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
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- 2021
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7. Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface
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Cesar A. López, Timothy Travers, Klaas M. Pos, Helen I. Zgurskaya, and S. Gnanakaran
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Medicine ,Science - Abstract
Abstract Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm that the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.
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- 2017
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8. Transport of lipophilic carboxylates is mediated by transmembrane helix 2 in multidrug transporter AcrB
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Christine Oswald, Heng-Keat Tam, and Klaas M. Pos
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Science - Abstract
The AcrB module of the AcrAB-TolC multidrug efflux pump sequesters drugs from the periplasm and outer leaflet of the inner membrane. Here, Oswaldet al. provide evidence that lipophilic carboxylated substrates bind to a groove between transmembrane helices TM1 and TM2, for further transport by an upward movement of TM2.
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- 2016
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9. Tripartite assembly of RND multidrug efflux pumps
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Laetitia Daury, François Orange, Jean-Christophe Taveau, Alice Verchère, Laura Monlezun, Céline Gounou, Ravi K. R. Marreddy, Martin Picard, Isabelle Broutin, Klaas M. Pos, and Olivier Lambert
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Science - Abstract
Tripartite efflux systems consist of inner membrane, outer membrane and periplasmic components. Here, Daury et al. reconstitute native versions of RND transporters in nanodiscs and present projection structures emphasizing the role of the periplasmic adaptor in linking the inner and outer membrane proteins.
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- 2016
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10. Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters
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Anja Roden, Melanie K. Engelin, Klaas M. Pos, and Eric R. Geertsma
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Clinical Biochemistry ,Molecular Biology ,Biochemistry - Abstract
Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.
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- 2023
11. Unidirectional mannitol synthesis of
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Heng-Keat, Tam, Patricia, König, Stephanie, Himpich, Ngoc Dinh, Ngu, Rupert, Abele, Volker, Müller, and Klaas M, Pos
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Acinetobacter baumannii ,Protein Subunits ,Osmotic Pressure ,Helix-Loop-Helix Motifs ,Mannitol ,Protein Multimerization ,Salt Stress ,Sugar Alcohol Dehydrogenases - Abstract
Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix–loop–helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.
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- 2022
12. Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters
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Jochen Wilhelm, Klaas M. Pos, Wuen Ee Foong, and H.K. Tam
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Acinetobacter baumannii ,Microbiology (medical) ,Tetracycline ,Microbial Sensitivity Tests ,Tigecycline ,medicine.disease_cause ,Microbiology ,Bacterial Proteins ,Drug Resistance, Multiple, Bacterial ,Escherichia coli ,polycyclic compounds ,medicine ,Pharmacology (medical) ,Pharmacology ,biology ,Membrane transport protein ,Chemistry ,Minocycline ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Major facilitator superfamily ,Anti-Bacterial Agents ,Infectious Diseases ,biology.protein ,Efflux ,Cell Division ,medicine.drug - Abstract
Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.
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- 2020
13. Binding and Transport of Carboxylated Drugs by the Multidrug Transporter AcrB
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Andrea Herrmann, H.K. Tam, Klaas M. Pos, Wuen-Ee Foong, Giuliano Malloci, Paolo Ruggerone, Viveka Nand Malviya, and Attilio Vittorio Vargiu
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Molecular model ,Protein Conformation ,Microbial Sensitivity Tests ,Molecular Dynamics Simulation ,medicine.disease_cause ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Escherichia coli ,medicine ,Binding site ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Chemistry ,Escherichia coli Proteins ,Transporter ,Periplasmic space ,Models, Theoretical ,Transmembrane domain ,Membrane protein ,Chromatography, Gel ,Biophysics ,Efflux ,Multidrug Resistance-Associated Proteins ,030217 neurology & neurosurgery - Abstract
AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2. In this article, the transport route from the initial TM1/TM2 groove binding site toward the deep binding pocket located in the periplasmic part has been addressed via molecular modeling studies followed by functional and structural characterization of several AcrB variants. We propose that membrane-embedded drugs bind initially to the TM1/TM2 groove, are oriented by the AcrB PN2 subdomain, and are subsequently transported via a PN2/PC1 interface pathway directly toward the deep binding pocket. Our work emphasizes the exploitation of multiple transport pathways by AcrB tuned to substrate physicochemical properties related to the polyspecificity of the pump.
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- 2020
14. Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria
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Ilyas Alav, Martin Picard, Vassiliy N. Bavro, Miriam S. Kuth, Jessica Kobylka, Jessica M A Blair, Klaas M. Pos, Membrane Transport Machinerie, Goethe-University Frankfurt am Main, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)
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Gram-negative bacteria ,Protein Conformation ,[SDV]Life Sciences [q-bio] ,Computational biology ,Review ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Structure-Activity Relationship ,Gram-Negative Bacteria ,Inner membrane ,Type I Secretion Systems ,biology ,010405 organic chemistry ,Chemistry ,Signal transducing adaptor protein ,Lipid bilayer fusion ,Membrane Transport Proteins ,General Chemistry ,Periplasmic space ,biology.organism_classification ,0104 chemical sciences ,Structural biology ,ATP-Binding Cassette Transporters ,Efflux ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
International audience; Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal componentsthe energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS-and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.
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- 2021
15. AcrB: a mean, keen, drug efflux machine
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Jessica Kobylka, Klaas M. Pos, Miriam S. Kuth, Eric R. Geertsma, and Reinke T. Müller
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Antiporter ,Protomer ,medicine.disease_cause ,Efflux pump complex ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Antibiotic resistance ,History and Philosophy of Science ,Drug Resistance, Multiple, Bacterial ,ddc:570 ,medicine ,Animals ,Humans ,Inner membrane ,Escherichia coli ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Chemistry ,Escherichia coli Proteins ,General Neuroscience ,Anti-Bacterial Agents ,Protein Structure, Tertiary ,Biophysics ,Efflux ,Multidrug Resistance-Associated Proteins ,Bacterial outer membrane - Abstract
Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.
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- 2019
16. Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB
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Thomas Eicher, Markus A Seeger, Claudio Anselmi, Wenchang Zhou, Lorenz Brandstätter, François Verrey, Kay Diederichs, José D Faraldo-Gómez, and Klaas M Pos
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antibiotic resistance ,efflux pump ,drug transport ,H+ transport ,mechanistic coupling ,drug resistance ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system.
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- 2014
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17. Antimicrobial Sensitivity Assay for
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Emanuele, Marine and Klaas M, Pos
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Methods Article - Abstract
Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of treatments. In order to make this approach feasible more has to be known about the ability of B. bacteriovorus to resist antibiotics itself. Standard assays to determine the minimum inhibitory concentration (MIC) are not suitable for B. bacteriovorus, since the small size of this bacterium (0.25-0.35 by 0.5-2 μm) prevents scattering at OD(600). Since these predatory bacteria require larger prey bacteria for growth (e.g., E. coli dimensions are 1 by 1-2 μm), the basis for the antimicrobial sensitivity assay described here is the reduction of the OD(600) caused by prey lysis during growth. Previous studies on predatory bacteria resistance to antimicrobials employed methods that did not allow a direct comparison of antimicrobial resistance levels to those of other bacterial species. Here, we describe a procedure to determine B. bacteriovorus sensitivity to antimicrobials which can be compared to a reference organism tested as close as possible to the same experimental conditions. Briefly, minimal inhibitory concentration (MIC) values of B. bacteriovorus are determined by measuring the reduction in absorbance at 600 nm of mixed predator/prey cultures in presence and absence of different antimicrobial concentrations. Of note, this method can be modified to obtain antimicrobial MIC values of other predatory bacteria, using different conditions, prey bacteria and/or antimicrobials.
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- 2020
18. Structural characterization of the EmrAB-TolC efflux complex from E. coli
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Selena Đorđević-Marquardt, Olivier Lambert, Alina Ornik-Cha, Klaas M. Pos, Mélanie Berbon, Jean-Christophe Taveau, Jean-William Dupuy, Marion Decossas, Narek Yousefian, Sylvie Poussard, Laetitia Daury, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Goethe-University Frankfurt am Main, Plateforme Protéome [Bordeaux], Centre Génomique Fonctionnelle Bordeaux [Bordeaux] (CGFB), and Institut Polytechnique de Bordeaux-Université de Bordeaux Ségalen [Bordeaux 2]-Institut Polytechnique de Bordeaux-Université de Bordeaux Ségalen [Bordeaux 2]
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[SDV]Life Sciences [q-bio] ,Biophysics ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,medicine ,Escherichia coli ,Inner membrane ,Protein Structure, Quaternary ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Chemistry ,Escherichia coli Proteins ,Signal transducing adaptor protein ,Membrane Proteins ,Membrane Transport Proteins ,Cell Biology ,Periplasmic space ,Membrane transport ,biochemical phenomena, metabolism, and nutrition ,Major facilitator superfamily ,Multiprotein Complexes ,bacteria ,Efflux ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
International audience; Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.
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- 2020
19. A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance
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Emanuele Marine, Carey Lambert, Renee Elizabeth Sockett, Klaas M. Pos, and David S. Milner
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0301 basic medicine ,medicine.drug_class ,Antibiotic sensitivity ,Antibiotics ,lcsh:Medicine ,Drug resistance ,Microbial Sensitivity Tests ,Trimethoprim ,Article ,antimicrobials ,Microbiology ,Bdellovibrio ,03 medical and health sciences ,enzyme mechanisms ,Dihydrofolate reductase ,Antibiosis ,Drug Resistance, Bacterial ,Gram-Negative Bacteria ,medicine ,lcsh:Science ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,lcsh:R ,Trimethoprim Resistance ,Bdellovibrio bacteriovorus ,3. Good health ,Anti-Bacterial Agents ,Multiple drug resistance ,030104 developmental biology ,biology.protein ,lcsh:Q ,medicine.drug - Abstract
Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).
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- 2020
20. A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter
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Eitan Bibi, Paolo Ruggerone, Klaas M. Pos, Michal Perach, Venkata Krishnan Ramaswamy, Elia Zomot, Eliane Hadas Yardeni, Giuliano Malloci, Attilio Vittorio Vargiu, H.K. Tam, and Yan, Nieng
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Models, Molecular ,0301 basic medicine ,Cytoplasm ,Mutant ,Crystallography, X-Ray ,medicine.disease_cause ,01 natural sciences ,Protein Structure, Secondary ,Substrate Specificity ,03 medical and health sciences ,Structural Biology ,0103 physical sciences ,Escherichia coli ,medicine ,Molecular Biology ,Binding Sites ,010304 chemical physics ,Chemistry ,Escherichia coli Proteins ,Membrane Transport Proteins ,Transporter ,Major facilitator superfamily ,Transport protein ,Molecular Docking Simulation ,Multiple drug resistance ,030104 developmental biology ,ddc:540 ,Mutation ,Biophysics ,Efflux ,Protein Binding - Abstract
Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.
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- 2018
21. Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump
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Klaas M. Pos, Reinke T. Müller, Joshua L. Phillips, Hi-jea Cha, Timothy Travers, and Sandrasegaram Gnanakaran
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Stereochemistry ,030106 microbiology ,Phenylalanine ,Microbial Sensitivity Tests ,Protomer ,03 medical and health sciences ,Structural Biology ,Drug Resistance, Multiple, Bacterial ,Switching loop ,Escherichia coli ,Proline ,Molecular Biology ,Binding Sites ,Chemistry ,Escherichia coli Proteins ,Substrate (chemistry) ,Biological Transport ,Anti-Bacterial Agents ,Loop (topology) ,030104 developmental biology ,Glycine ,Efflux ,Multidrug Resistance-Associated Proteins ,Protein Binding - Abstract
The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.
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- 2017
22. Identification of the novel class D v-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus
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Sara Riedel-Christ, Ingo Ebersberger, Ewgenij Proschak, Bardya Djahanschiri, Stephan Göttig, Jan S. Kramer, Sonali Wohra, Klaas M. Pos, Manuela Tietgen, Ulrich Nübel, Matthias Steglich, Aitor Gonzaga, Paul G. Higgins, Marko Weidensdorfer, Steffen Brunst, and Publica
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0301 basic medicine ,Microbiology (medical) ,Acinetobacter baumannii ,Models, Molecular ,Carbapenem ,Imipenem ,medicine.drug_class ,Protein Conformation ,030106 microbiology ,Antibiotics ,Microbial Sensitivity Tests ,Moths ,medicine.disease_cause ,DNA sequencing ,beta-Lactamases ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Drug Resistance, Bacterial ,polycyclic compounds ,medicine ,Animals ,Humans ,Pharmacology (medical) ,030212 general & internal medicine ,Amino Acid Sequence ,Acinetobacter calcoaceticus ,Escherichia coli ,Pharmacology ,biology ,Whole Genome Sequencing ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,3. Good health ,Anti-Bacterial Agents ,Galleria mellonella ,Infectious Diseases ,Carbapenems ,Larva ,bacteria ,medicine.drug ,Acinetobacter Infections - Abstract
Objectives: The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D v-lactamase (CHDL) OXA-679. Methods: Identification of the species and v-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. Results: Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. Conclusions: We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.
- Published
- 2019
23. The chloramphenicol/H+ antiporter CraA of Acinetobacter baumannii AYE reveals a broad substrate specificity
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Jan J Crames, Klaas M. Pos, Beate Averhoff, Wuen Ee Foong, and H.K. Tam
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0301 basic medicine ,Microbiology (medical) ,Florfenicol ,Acinetobacter baumannii ,Models, Molecular ,Protein Conformation ,030106 microbiology ,medicine.disease_cause ,Antiporters ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacterial Proteins ,Drug Resistance, Bacterial ,medicine ,Pharmacology (medical) ,030212 general & internal medicine ,Amino Acid Sequence ,Cloning, Molecular ,Escherichia coli ,Pharmacology ,Dequalinium ,biology ,Membrane transport protein ,Chloramphenicol ,Biological Transport ,Sequence Analysis, DNA ,biology.organism_classification ,Thiamphenicol ,Molecular biology ,Major facilitator superfamily ,Anti-Bacterial Agents ,Infectious Diseases ,chemistry ,biology.protein ,medicine.drug ,Acinetobacter Infections ,Hydrogen - Abstract
Objectives To identify major facilitator superfamily (MFS)-type chloramphenicol transporters of Acinetobacter baumannii AYE, to characterize its substrate specificity and identify CraA substrate and H+ binding sites. Methods Five ORFs predicted to encode chloramphenicol transporters were heterologously expressed in Escherichia coli and their substrate specificity was determined by drug susceptibility assays on solid agar medium. CraA transport properties were determined via whole cell fluorescence experiments using ethidium and dequalinium. ACMA quenching was used to characterize the H+/drug antiport process in everted membrane vesicles. The function of CraA in A. baumannii was determined by drug susceptibility assay using A. baumannii ATCC 19606 ΔcraA. Results CraA, ABAYE0913 and CmlA5 are functionally active when overproduced in E. coli. ABAYE0913 conferred resistance to florfenicol and benzalkonium, CmlA5 conferred resistance to chloramphenicol and thiamphenicol, and craA expression resulted in resistance to chloramphenicol, thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C and TPP+. Cell expressing craA_E38A showed no resistance to all tested drugs, implying that Glu-38 is involved in the binding of drugs and/or protons. Functional assays indicated that substitution of Asp-46 to Ala resulted in severe susceptibility to cationic drugs, chloramphenicol and thiamphenicol. In contrast, Glu-338 is important for the recognition of chloramphenicol, florfenicol, chlorhexidine and dequalinium. Conclusions This study suggests that CraA has a broad substrate specificity, similar to that of E. coli MdfA. However, due to the presence of three charged residues in the transmembrane region conferring different susceptibility profiles upon substitution to Ala, we postulate that CraA has a different substrate recognition mode compared with MdfA.
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- 2018
24. Identification and characterization of carbapenem binding sites within the RND-transporter AcrB
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Alessio Atzori, Attilio Vittorio Vargiu, Paolo Ruggerone, Giuliano Malloci, Klaas M. Pos, Jürg Dreier, and Viveka Nand Malviya
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Imipenem ,Carbapenem ,Biophysics ,Molecular Conformation ,Microbial Sensitivity Tests ,Molecular Dynamics Simulation ,Biochemistry ,Meropenem ,03 medical and health sciences ,Drug Resistance, Multiple, Bacterial ,polycyclic compounds ,medicine ,Escherichia coli ,Binding site ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Calorimetry, Differential Scanning ,030306 microbiology ,Chemistry ,Escherichia coli Proteins ,Rational design ,Isothermal titration calorimetry ,Transporter ,Biological Transport ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Anti-Bacterial Agents ,Molecular Docking Simulation ,Carbapenems ,Thermodynamics ,Efflux ,Multidrug Resistance-Associated Proteins ,medicine.drug ,Bacterial Outer Membrane Proteins ,Protein Binding - Abstract
Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.
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- 2018
25. Multidrug efflux pumps: structure, function and regulation
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Klaas M. Pos, Dijun Du, Hendrik W. van Veen, Laura J. V. Piddock, Xuan Wang-Kan, Arthur Neuberger, and Ben F. Luisi
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0301 basic medicine ,030106 microbiology ,Virulence ,Drug resistance ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Antibiotic resistance ,Bacterial Proteins ,Drug Resistance, Bacterial ,Gram-Negative Bacteria ,medicine ,General Immunology and Microbiology ,biology ,Structure function ,Membrane Transport Proteins ,Pathogenic bacteria ,Bacterial pathogenesis ,biology.organism_classification ,Drug Resistance, Multiple ,Cell biology ,030104 developmental biology ,Infectious Diseases ,ATP-Binding Cassette Transporters ,Efflux ,Gram-Negative Bacterial Infections ,Bacteria - Abstract
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.
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- 2018
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26. Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface
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Sandrasegaram Gnanakaran, Klaas M. Pos, Timothy Travers, Helen I. Zgurskaya, and Cesar A. Lopez
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Science ,Context (language use) ,Molecular Dynamics Simulation ,medicine.disease_cause ,Article ,Bacterial cell structure ,Structure-Activity Relationship ,03 medical and health sciences ,medicine ,Inner membrane ,Multidisciplinary ,Pseudomonas aeruginosa ,Chemistry ,Genetic Variation ,Membrane Transport Proteins ,Periplasmic space ,030104 developmental biology ,Membrane ,Biophysics ,Medicine ,Efflux ,Protein Multimerization ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins ,Protein Binding - Abstract
Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm that the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.
- Published
- 2017
27. High-Resolution Crystallographic Analysis of AcrB Using Designed Ankyrin Repeat Proteins (DARPins)
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Heng Keat, Tam, Viveka Nand, Malviya, and Klaas M, Pos
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Models, Molecular ,Protein Conformation ,Escherichia coli Proteins ,Multiprotein Complexes ,Chromatography, Gel ,Escherichia coli ,Multidrug Resistance-Associated Proteins ,Crystallography, X-Ray ,Chromatography, Affinity ,Ankyrin Repeat ,Protein Binding - Abstract
X-ray crystallography is still the most prominent technique in use to decipher the 3D structures of membrane proteins. For successful crystallization, sample quality is the most important parameter that should be addressed. In almost every case, highly pure, monodisperse, and stable protein sample is a prerequisite. Vapor diffusion is in general the method of choice for obtaining crystals. Here, we discuss a detailed protocol for overproduction and purification of the inner-membrane multidrug transporter AcrB and of DARPins, which are used for crystallization of the AcrB/DARPin complex, resulting in high-resolution diffraction and subsequent structure determination.
- Published
- 2017
28. High-Resolution Crystallographic Analysis of AcrB Using Designed Ankyrin Repeat Proteins (DARPins)
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Heng Keat Tam, Klaas M. Pos, and Viveka Nand Malviya
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0301 basic medicine ,Materials science ,High resolution ,law.invention ,Sample quality ,03 medical and health sciences ,Crystallography ,030104 developmental biology ,DARPin ,Membrane protein ,law ,Ankyrin repeat ,Crystallization ,Multidrug transporter - Abstract
X-ray crystallography is still the most prominent technique in use to decipher the 3D structures of membrane proteins. For successful crystallization, sample quality is the most important parameter that should be addressed. In almost every case, highly pure, monodisperse, and stable protein sample is a prerequisite. Vapor diffusion is in general the method of choice for obtaining crystals. Here, we discuss a detailed protocol for overproduction and purification of the inner-membrane multidrug transporter AcrB and of DARPins, which are used for crystallization of the AcrB/DARPin complex, resulting in high-resolution diffraction and subsequent structure determination.
- Published
- 2017
29. BGA66 and BGA71 facilitate complement resistance ofBorrelia bavariensisby inhibiting assembly of the membrane attack complex
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Volker Fingerle, Reinhard Wallich, Christine Skerka, Claudia Hammerschmidt, Klaas M. Pos, Yvonne Klevenhaus, Peter F. Zipfel, Peter Kraiczy, Teresia Hallström, and Arno Koenigs
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0301 basic medicine ,Complement component 2 ,Complement receptor ,Biology ,Microbiology ,Complement factor B ,Virology ,Cell biology ,Complement system ,03 medical and health sciences ,Classical complement pathway ,030104 developmental biology ,Factor H ,biology.protein ,Complement membrane attack complex ,Molecular Biology ,Complement control protein - Abstract
Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.
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- 2015
30. Approved Drugs Containing Thiols as Inhibitors of Metallo-β-lactamases: Strategy To Combat Multidrug-Resistant Bacteria
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Müller Hf, Stephan Göttig, Ewgenij Proschak, Cuesta-Bernal J, Denia Frank, Arno Koenigs, Klaas M. Pos, Franca-Maria Klingler, Hanno Sjuts, Jasmin El-Delik, Thomas A. Wichelhaus, and Denys Pogoryelov
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Models, Molecular ,Imipenem ,medicine.drug_class ,Antibiotics ,Antimicrobial susceptibility ,Pharmacology ,Crystallography, X-Ray ,beta-Lactams ,beta-Lactam Resistance ,beta-Lactamases ,Metallo β lactamase ,Drug Resistance, Multiple, Bacterial ,Drug Discovery ,Escherichia coli ,polycyclic compounds ,Screening method ,medicine ,Humans ,Pseudomonas Infections ,Sulfhydryl Compounds ,Escherichia coli Infections ,chemistry.chemical_classification ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Anti-Bacterial Agents ,Klebsiella Infections ,Klebsiella pneumoniae ,Enzyme ,Multidrug resistant bacteria ,chemistry ,Pseudomonas aeruginosa ,Molecular Medicine ,beta-Lactamase Inhibitors ,medicine.drug - Abstract
Resistance to β-lactam antibiotics can be mediated by metallo-β-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of β-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1, and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods, we identified drugs that restored the activity of imipenem.
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- 2015
31. Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria
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Jean-Marie Pagès, Klaas M. Pos, Matthieu Réfrégiers, Muriel Masi, Transporteurs membranaires, chimioresistance et drug-design (TMCD2), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Membrane Transport Machinerie, Goethe-University Frankfurt am Main, Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115525, resources which are composed of financial contribution from the European Union’s seventh framework program (FP/2007-2013) and EFPIA companies in kind contributions, and European Project: 115525,Translocation
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0301 basic medicine ,Microbiology (medical) ,Drug ,Gram-negative bacteria ,Membrane permeability ,medicine.drug_class ,media_common.quotation_subject ,[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] ,030106 microbiology ,Immunology ,Antibiotics ,Biological Transport, Active ,translocation ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,Permeability ,antibiotics ,Diffusion ,resistance ,03 medical and health sciences ,Antibiotic resistance ,Cell Wall ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Gram-Negative Bacteria ,Genetics ,medicine ,media_common ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Cell Membrane ,Membrane Transport Proteins ,Cell Biology ,Antimicrobial ,biology.organism_classification ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Anti-Bacterial Agents ,3. Good health ,030104 developmental biology ,Efflux ,Bacteria - Abstract
Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are characterized by low permeability and often contain drug efflux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or efflux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identified clear correlations between efflux activity and drug diffusion and accumulation in both susceptible and resistant strains, and have also clarified how certain chemical structures can affect drug entry and residence time within the cell. In this PERSPECTIVE, we discuss the biological underpinnings of drug permeability and export using several prototypical influx and efflux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid efforts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics. A Perspective on unravelling the mechanisms of antibiotic penetration and efflux in Gram-negative bacteria.
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- 2017
32. Cytochrome c oxidase biogenesis and metallochaperone interactions : steps in the assembly pathway of a bacterial complex
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Sonja Schimo, Klaas M. Pos, Bernd Ludwig, Ilka Wittig, and Giuffrè, Alessandro
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0301 basic medicine ,Scaffold protein ,lcsh:Medicine ,Biochemistry ,Polyacrylamide Gel Electrophoresis ,Mass Spectrometry ,chemistry.chemical_compound ,Blue Native Polyacrylamide Gel Electrophoresis ,Post-Translational Modification ,lcsh:Science ,Enzyme Chemistry ,Heme ,Energy-Producing Organelles ,Gel Electrophoresis ,Oxidase test ,Multidisciplinary ,biology ,Mitochondria ,Precipitation Techniques ,Metallochaperones ,Electrophoresis, Polyacrylamide Gel ,Cellular Structures and Organelles ,Protein Binding ,Research Article ,Protein subunit ,Bioenergetics ,Biosynthesis ,Research and Analysis Methods ,Protein–protein interaction ,Electron Transport Complex IV ,03 medical and health sciences ,Electrophoretic Techniques ,ddc:570 ,Immunoprecipitation ,Protein Interactions ,Paracoccus denitrificans ,030102 biochemistry & molecular biology ,lcsh:R ,Biology and Life Sciences ,Proteins ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,chemistry ,Chaperone (protein) ,biology.protein ,Enzymology ,Cofactors (Biochemistry) ,lcsh:Q ,Biogenesis - Abstract
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.
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- 2017
33. Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP
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Rupert Abele, Ahmad Reza Mehdipour, Vincent Olieric, Thomas M. Tomasiak, Klaas M. Pos, Anne Nöll, Stefan Brüchert, Robert M. Stroud, Christoph Thomas, Kay Diederichs, Meitian Wang, Gerhard Hummer, Robert Tampé, Benesh Joseph, Tina Zollmann, Valentina Herbring, and Katja Barth
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0301 basic medicine ,Models, Molecular ,Protein Conformation ,ATP-binding cassette transporter ,Biology ,Catalysis ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,ddc:570 ,Humans ,ATP-binding domain of ABC transporters ,ABC transporter, conformational dynamics, membrane proteins, peptide transport, transporter associated with antigen processing ,Multidisciplinary ,Binding Sites ,Antigen processing ,Thermus thermophilus ,Transporter associated with antigen processing ,biology.organism_classification ,Drug Resistance, Multiple ,3. Good health ,Transport protein ,030104 developmental biology ,Membrane protein ,Biochemistry ,PNAS Plus ,Peptide transport ,ATP-Binding Cassette Transporters ,030217 neurology & neurosurgery - Abstract
ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular. published
- Published
- 2017
34. Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt
- Author
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Björn Windshügel, Satya Prathyusha Bhamidimarri, Klaas M. Pos, Ravi K. R. Marreddy, Mathias Winterhalter, L. Benier, Mark Brönstrup, U. Bilitewski, Philip Gribbon, A. Gilardi, and Publica
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0301 basic medicine ,Biophysics ,Gating ,Biochemistry ,Biophysical Phenomena ,03 medical and health sciences ,Minimum inhibitory concentration ,Drug Resistance, Multiple, Bacterial ,Escherichia coli ,Channel blocker ,Surface plasmon resonance ,Molecular Biology ,Chemistry ,Escherichia coli Proteins ,Membrane Transport Proteins ,Cobalt ,Gene Expression Regulation, Bacterial ,Surface Plasmon Resonance ,biochemical phenomena, metabolism, and nutrition ,Small molecule ,Dissociation constant ,030104 developmental biology ,bacteria ,Efflux ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
Background The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. Methods We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. Results TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. Conclusions TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. General significance TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.
- Published
- 2017
35. Bacterial efflux transporters in the limelight
- Author
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Klaas M. Pos
- Subjects
Limelight ,Bacteria ,Bacterial Proteins ,Biochemistry ,law ,Membrane Transport Proteins ,General Medicine ,Biology ,Molecular Biology ,Microbiology ,law.invention ,Efflux transporters - Published
- 2018
36. Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR
- Author
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Klaas M. Pos, Andrea Lakatos, Johanna Becker-Baldus, Clemens Glaubitz, and Yean Sin Ong
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education.field_of_study ,Molecular Structure ,Stereochemistry ,Escherichia coli Proteins ,Dimer ,Antiporter ,Population ,Trityl Compounds ,General Chemistry ,Ligand (biochemistry) ,Biochemistry ,Antiporters ,Catalysis ,chemistry.chemical_compound ,Transmembrane domain ,Onium Compounds ,Organophosphorus Compounds ,Colloid and Surface Chemistry ,chemistry ,Solid-state nuclear magnetic resonance ,Drug Resistance, Multiple, Bacterial ,Efflux ,education ,Nuclear Magnetic Resonance, Biomolecular ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP(+) and MTP(+) to EmrE reconstituted into 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes by (31)P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between (13)C-labeled TPP(+) and key residues within the EmrE dimer has been probed by through-space (13)C-(13)C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such as TPP(+) and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection.
- Published
- 2013
37. The Outer Membrane TolC-like Channel HgdD Is Part of Tripartite Resistance-Nodulation-Cell Division (RND) Efflux Systems Conferring Multiple-drug Resistance in the Cyanobacterium Anabaena sp. PCC7120
- Author
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Iryna Lytvynenko, Klaas M. Pos, Oliver Mirus, Alexander Hahn, Enrico Schleiff, and Mara Stevanovic
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Mutant ,Plant Biology ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Drug Resistance, Multiple, Bacterial ,Ethidium ,medicine ,Inner membrane ,Enzyme Inhibitors ,Molecular Biology ,Escherichia coli ,biology ,Membrane fusion protein ,Anabaena ,Gene Expression Regulation, Bacterial ,Cell Biology ,biology.organism_classification ,Anti-Bacterial Agents ,Erythromycin ,Mutagenesis, Insertional ,chemistry ,bacteria ,Efflux ,Carrier Proteins ,Bacterial outer membrane ,Ethidium bromide ,Bacterial Outer Membrane Proteins - Abstract
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
- Published
- 2013
38. RND efflux pumps: structural information translated into function and inhibition mechanisms
- Author
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Klaas M. Pos, Paolo Ruggerone, Attilio Vittorio Vargiu, and Satoshi Murakami
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Proton translocation ,Models, Molecular ,Protein Conformation ,Antiporter ,Escherichia coli Proteins ,Lipoproteins ,Cell Membrane ,Membrane Transport Proteins ,SUPERFAMILY ,General Medicine ,Drug resistance ,Biology ,Cell biology ,Anti-Bacterial Agents ,Antibiotic resistance ,Protein structure ,Biochemistry ,Bacterial Proteins ,Drug Resistance, Multiple, Bacterial ,Drug Discovery ,Gram-Negative Bacteria ,Efflux ,Multidrug Resistance-Associated Proteins ,Function (biology) ,Bacterial Outer Membrane Proteins - Abstract
Efflux pumps of the Resistance Nodulation Division (RND) superfamily play a major role in the intrinsic and acquired resistance of Gram-negative pathogens to antibiotics. Moreover, they are largely responsible for multi-drug resistance (MDR) phenomena in these bacteria. The last decade has seen a sharp increase in the number of experimental and computational studies aimed at understanding their functional mechanisms. Most of these studies focused on the RND drug/proton antiporter AcrB, part of the AcrAB-TolC efflux pump actively recognizing and expelling noxious agents from the interior of bacteria. These studies have been focused on the dynamical interactions between AcrB and its substrates and inhibitors, on the details of the proton translocation mechanisms, and on the way AcrB assembles with protein partners to build up a functional pump. In this review we summarize these advances focusing on the role of AcrB.
- Published
- 2013
39. Editorial : bad bugs in the XXIst century: resistance mediated by multi-drug efflux pumps in gram-negative bacteria
- Author
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Klaas M. Pos, Keith Poole, Attilio Vittorio Vargiu, Hiroshi Nikaido, and Nishino, Kunihiko
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0301 basic medicine ,Microbiology (medical) ,Klebsiella ,Gram-negative bacteria ,antibiotic resistance ,medicine.drug_class ,efflux pumps ,Antibiotics ,lcsh:QR1-502 ,Drug resistance ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,Antibiotic resistance ,multi-drug-resistant pathogens ,ddc:570 ,medicine ,bacterial resistance mechanisms ,biology ,biology.organism_classification ,Antimicrobial ,3. Good health ,Multiple drug resistance ,Editorial ,030104 developmental biology ,superbugs ,Efflux - Abstract
The discovery of antibiotics represented a key milestone in the history of medicine. However, with the rise of these life-saving drugs came the awareness that bacteria deploy defense mechanisms to resist these antibiotics, and they are good at it. Today, we appear at a crossroads between discovery of new potent drugs and omni-resistant superbugs. Moreover, the misuse of antibiotics in different industries has increased the rate of resistance development by providing permanent selective pressure and, subsequently, enrichment of multidrug resistant pathogens. As a result, antimicrobial resistance has now become an urgent threat to public health worldwide (http:// www.who.int/drugresistance/documents/surveillancereport/en/). The development of multidrug resistance (MDR) in an increasing number of pathogens, including Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Burkholderia, and other Gram-negative bacteria is a serious issue. Membrane efflux pump complexes of the Resistance-Nodulation-Division (RND) superfamily play a key role in the development of MDR in these bacteria. These pumps, together with other transporters, contribute to intrinsic and acquired resistance of bacteria toward most, if not all, of the compounds available in our antimicrobial arsenal. Given the enormous drug polyspecificity of MDR efflux pumps, studies on their mechanism of action are extremely challenging, and this has negatively impacted both on the development of new antibiotics that are able to evade these efflux pumps and on the design of pump inhibitors. The collection of articles in this eBook, published as a Research Topic in Frontiers in Microbiology, section of Antimicrobials, Resistance, and Chemotherapy, aims to update the reader about the latest advances on the structure and function of RND efflux transporters, their roles in the overall multidrug resistance phenotype of Gram-negative pathogens, and on the strategies to inhibit their activities. ...
- Published
- 2016
40. Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives
- Author
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Steven M. Kwasny, Timothy J. Opperman, Xiaoyuan Ding, Hanno Sjuts, Terry L. Bowlin, Alina R. Ornik, Attilio Vittorio Vargiu, Son T. Nguyen, Paolo Ruggerone, Hiroshi Nikaido, Hong-Suk Kim, and Klaas M. Pos
- Subjects
0301 basic medicine ,Models, Molecular ,Cell division ,Pyridines ,Drug Resistance ,medicine.disease_cause ,Crystallography, X-Ray ,Models ,Drug Resistance, Multiple, Bacterial ,Drug Discovery ,efflux pump inhibitors ,Crystallography ,Multidisciplinary ,Escherichia coli Proteins ,Bacterial ,Biological Sciences ,Anti-Bacterial Agents ,Infectious Diseases ,molecular dynamics simulation ,Biochemistry ,5.1 Pharmaceuticals ,Efflux ,Multidrug Resistance-Associated Proteins ,Infection ,Multiple ,Hydrophobic and Hydrophilic Interactions ,Protein Structure ,Stereochemistry ,030106 microbiology ,Drug design ,Biology ,Molecular Dynamics Simulation ,Vaccine Related ,03 medical and health sciences ,multidrug resistance ,Biodefense ,medicine ,Escherichia coli ,Humans ,Antibacterial drug ,RND efflux transporters ,X-ray crystallography ,Pyrans ,Binding Sites ,Prevention ,Molecular ,Periplasmic space ,biology.organism_classification ,Protein Structure, Tertiary ,Multiple drug resistance ,Emerging Infectious Diseases ,030104 developmental biology ,ddc:000 ,X-Ray ,Antimicrobial Resistance ,Tertiary ,Bacteria - Abstract
Proceedings of the National Academy of Sciences of the United States of America 113(13), 3509 - 3514(2016). doi:10.1073/pnas.1602472113, The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens., Published by National Acad. of Sciences, Washington, DC
- Published
- 2016
41. Tripartite assembly of RND multidrug efflux pumps
- Author
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Isabelle Broutin, Laura Monlezun, Olivier Lambert, Ravi K. R. Marreddy, Martin Picard, Alice Verchère, Klaas M. Pos, François Orange, Jean-Christophe Taveau, Laetitia Daury, Céline Gounou, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratoire de chimie moléculaire et thioorganique (LCMT), Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU), Imagerie Moléculaire et Nanobiotechnologies - Institut Européen de Chimie et Biologie (IECB), Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Membrane Transport Machinerie, Goethe-University Frankfurt am Main, Ecole Nationale Vétérinaire Agroalimentaire et de l'Alimentation Nantes Atlantique (ONIRIS), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Centre National de la Recherche Scientifique (CNRS)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Le Havre Normandie (ULH), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Science ,[SDV]Life Sciences [q-bio] ,Lipoproteins ,030106 microbiology ,General Physics and Astronomy ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Microscopy, Electron, Transmission ,Drug Resistance, Multiple, Bacterial ,Escherichia coli ,Inner membrane ,Outer membrane efflux proteins ,Nanodisc ,Multidisciplinary ,biology ,Membrane transport protein ,Escherichia coli Proteins ,Membrane Transport Proteins ,Signal transducing adaptor protein ,General Chemistry ,Periplasmic space ,Nanostructures ,Cell biology ,Native Polyacrylamide Gel Electrophoresis ,Multiprotein Complexes ,Pseudomonas aeruginosa ,biology.protein ,bacteria ,Multidrug Resistance-Associated Proteins ,Periplasmic Proteins ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components., Tripartite efflux systems consist of inner membrane, outer membrane and periplasmic components. Here, Daury et al. reconstitute native versions of RND transporters in nanodiscs and present projection structures emphasizing the role of the periplasmic adaptor in linking the inner and outer membrane proteins.
- Published
- 2016
42. Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop
- Author
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Thomas Eicher, Markus A. Seeger, Kay Diederichs, Lorenz Brandstätter, Jürgen A. Bohnert, Markus G. Grütter, Winfried V. Kern, Klaas M. Pos, Jasmin El-Delik, Hi-jea Cha, François Verrey, University of Zurich, and Pos, Klaas M
- Subjects
Models, Molecular ,Stereochemistry ,610 Medicine & health ,Minocycline ,Plasma protein binding ,Biology ,Protein Structure, Secondary ,10052 Institute of Physiology ,chemistry.chemical_compound ,Protein structure ,Antibiotics ,multidrug resistance ,ddc:570 ,10019 Department of Biochemistry ,Inner membrane ,Binding site ,1000 Multidisciplinary ,Binding Sites ,Multidisciplinary ,Escherichia coli Proteins ,Biological Transport ,Periplasmic space ,Biological Sciences ,drug efflux ,Transport protein ,Monomer ,Pharmaceutical Preparations ,chemistry ,DARPin ,Doxorubicin ,10076 Center for Integrative Human Physiology ,Biocatalysis ,570 Life sciences ,biology ,RND transporter ,Multidrug Resistance-Associated Proteins ,Protein Binding - Abstract
AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9–2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.
- Published
- 2012
43. Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS
- Author
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Klaas M. Pos, Winfried V. Kern, Christophe Briand, Lucie Sokolova, Lorenz Brandstätter, Bernd Brutschy, Hi-jea Cha, Jürgen A. Bohnert, Mihaela Cernescu, Thomas Eicher, and Markus A. Seeger
- Subjects
Electrophoresis ,Drug export ,Detergents ,Biophysics ,Trimer ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Mass Spectrometry ,Membrane protein complex ,Escherichia coli ,LILBID MS ,Inner membrane ,Micelles ,Phospholipids ,DARPins ,Ligand ,Chemistry ,Escherichia coli Proteins ,Electrophoresis, Capillary ,Complex formation ,AcrB ,Cell Biology ,Periplasmic space ,Ankyrin Repeat ,Crystallography ,DARPin ,Mutation ,Multidrug efflux ,Ankyrin repeat ,Chromatography, Thin Layer ,Multidrug Resistance-Associated Proteins ,Crystallization ,Bacterial outer membrane ,Dimerization ,Ultracentrifugation - Abstract
article i nfo The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/ DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1 kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.
- Published
- 2011
44. Author Correction: Multidrug efflux pumps: structure, function and regulation
- Author
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Laura J. V. Piddock, Klaas M. Pos, Ben F. Luisi, Arthur Neuberger, Hendrik W. van Veen, Dijun Du, and Xuan Wang-Kan
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0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Infectious Diseases ,General Immunology and Microbiology ,Published Erratum ,Structure function ,MEDLINE ,Library science ,Biology ,Microbiology - Abstract
In the version of this Review originally published, the author contributions of co-author Arthur Neuberger were incorrectly listed. The author contributions should have appeared as 'D.D., X.W.-K., A.N., H.W.v.V., K.M.P., L.J.V.P. and B.F.L. researched data for the article, made substantial contributions to discussions of the content, wrote the article, and reviewed and edited the manuscript before submission'. This has now been corrected in all versions of the Review. The authors apologize to readers for this error.
- Published
- 2018
45. The use of novel organic gels and hydrogels in protein crystallization
- Author
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Abel Moreno, Hong-Ting Lin, Zbigniew Pietras, Klaas M. Pos, Sachin Surade, Ben F. Luisi, and Orla Slattery
- Subjects
Aqueous solution ,food.ingredient ,Chemistry ,Polyvinyl alcohol ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,food ,Membrane protein ,Chemical engineering ,Thaumatin ,Self-healing hydrogels ,Organic chemistry ,Agar ,Lysozyme ,Protein crystallization - Abstract
The use of an organic solvent-based gel prepared from polyethylene oxide and a polyvinyl alcohol hydrogel for protein crystallization was investigated. The preparation, properties and application of the gels for protein crystallization are described, and the advantages and limitations of the approach are discussed. The gels are compared with agar, which is a popular aqueous gel used for protein crystallization. The growth behaviour and diffraction quality of crystals prepared in these gel media were evaluated for two model soluble proteins, thaumatin and lysozyme, and for two bacterial membrane proteins, TolC and AcrB.
- Published
- 2010
46. Drug transport mechanism of the AcrB efflux pump
- Author
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Klaas M. Pos
- Subjects
Models, Molecular ,Proton binding ,Protein Conformation ,Lipoproteins ,Biophysics ,Biology ,Models, Biological ,Biochemistry ,Analytical Chemistry ,Molecular Biology ,Ion transporter ,Ion Transport ,Membrane transport protein ,Escherichia coli Proteins ,Membrane Transport Proteins ,Periplasmic space ,Transmembrane protein ,Transmembrane domain ,biology.protein ,Efflux ,Multidrug Resistance-Associated Proteins ,Energy Metabolism ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude cytotoxic substances from the cell directly into the medium bypassing periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump that extrudes multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB, a member of the Resistance Nodulation cell Division (RND) family, is the major site for substrate recognition and energy transduction of the entire tripartite system. The drug/proton antiport processes in this secondary transporter are suggested to be spatially separated, a feature frequently observed for primary transporters like membrane-bound ATPases. The recently elucidated asymmetric structure of the AcrB trimer reveals three different monomer conformations proposed to represent consecutive states in a directional transport cycle. Each monomer shows a distinct tunnel system with entrances located at the boundary of the outer leaflet of the inner membrane and the periplasm through the periplasmic porter (pore) domain towards the funnel of the trimer and TolC. In one monomer a hydrophobic pocket is present which has been shown to bind the AcrB substrates minocyclin and doxorubicin. The energy conversion from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (D407/D408/K940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel drug transport model combines the alternate access pump mechanism with the rotating site catalysis of F(1)F(o) ATPase as originally postulated by Jardetzky and Boyer, respectively, and suggests a working hypothesis for the transport mechanism of RND transporters in general.
- Published
- 2009
47. Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis
- Author
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Stefanie Keis, Jonathan C. Gauntlett, Gregory M. Cook, Klaas M. Pos, Susanne Gebhard, and Janet M. Manson
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Operon ,Molecular Sequence Data ,Plasma protein binding ,Biology ,medicine.disease_cause ,Biochemistry ,DNA-binding protein ,chemistry.chemical_compound ,Bacitracin ,Enterococcus faecalis ,medicine ,Amino Acid Sequence ,Promoter Regions, Genetic ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Cell Membrane ,Promoter ,Gene Expression Regulation, Bacterial ,Cell Biology ,Molecular biology ,Transmembrane protein ,DNA-Binding Proteins ,chemistry ,Mutation ,ATP-Binding Cassette Transporters ,DNA ,Protein Binding - Abstract
BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.
- Published
- 2008
48. Molecular basis of polyspecificity of the Small Multidrug Resistance Efflux Pump AbeS from Acinetobacter baumannii
- Author
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Shlomo Brill, Klaas M. Pos, Iryna Lytvynenko, and Christine Oswald
- Subjects
0301 basic medicine ,Acinetobacter baumannii ,Models, Molecular ,Antiporter ,030106 microbiology ,Molecular Sequence Data ,Biology ,Antiporters ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Drug Resistance, Multiple, Bacterial ,Escherichia coli ,Humans ,Point Mutation ,Small multidrug resistance protein ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Escherichia coli Proteins ,Biological Transport ,Membrane transport ,biology.organism_classification ,Anti-Bacterial Agents ,Multiple drug resistance ,030104 developmental biology ,Biochemistry ,chemistry ,Acriflavine ,Efflux ,Sequence Alignment ,Acinetobacter Infections - Abstract
Secondary multidrug efflux transporters play a key role in the bacterial resistance phenotype. One of the major questions concerns the polyspecific recognition of substrates by these efflux pumps. To understand the molecular basis of this promiscuous recognition, we compared the substrate specificity of the well-studied Escherichia coli small multidrug resistance protein EmrE with that of the poorly studied Acinetobacter baumannii homologue AbeS. The latter drug/H(+) antiporter is a 109-amino-acid membrane protein with predicted four transmembrane helices. It effectively confers resistance toward ethidium, acriflavine and benzalkonium in an E. coli ΔemrEΔmdfA background. Purified AbeS and the substrate-specific hyperactive variant A16G bind tetraphenylphosphonium with nanomolar affinity and exhibit electrogenic transport of 1-methyl-4-phenylpyridinium after reconstitution into liposomes. A16G hyperactivity was apparent toward acriflavine and ethidium, resulting in 7- to 10-fold higher normalized IC50 values, respectively, but not toward substrates 1-methyl-4-phenylpyridinium and benzalkonium. Substitution of Y3 and A42 with Ala or Ser, respectively, also displayed a substrate-dependent phenotype, as these variants were strongly affected in their properties to confer resistance against acriflavine and ethidium, but not against benzalkonium. The size and planarity of the conjugated aromatic moieties appear to be a critical and subtle criterion for substrate recognition by these transporters. Rather moderate changes in the property of side chains postulated to be part of the substrate binding site result in a large phenotypical difference. These observations provide indications for the molecular basis of specificity within the binding pocket of polyspecific transporters.
- Published
- 2015
49. Structure, mechanism and cooperation of bacterial multidrug transporters
- Author
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Dijun, Du, Hendrik W, van Veen, Satoshi, Murakami, Klaas M, Pos, and Ben F, Luisi
- Subjects
Bacterial Proteins ,Cell Wall ,Protein Conformation ,Drug Resistance, Multiple, Bacterial ,Cell Membrane ,Gram-Negative Bacteria ,Membrane Transport Proteins ,ATP-Binding Cassette Transporters ,Anti-Bacterial Agents - Abstract
Cells from all domains of life encode energy-dependent trans-membrane transporters that can expel harmful substances including clinically applied therapeutic agents. As a collective body, these transporters perform as a super-system that confers tolerance to an enormous range of harmful compounds and consequently aid survival in hazardous environments. In the Gram-negative bacteria, some of these transporters serve as energy-transducing components of tripartite assemblies that actively efflux drugs and other harmful compounds, as well as deliver virulence agents across the entire cell envelope. We draw together recent structural and functional data to present the current models for the transport mechanisms for the main classes of multi-drug transporters and their higher-order assemblies.
- Published
- 2015
50. The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump
- Author
-
Klaas M. Pos and Reinke T. Müller
- Subjects
Models, Molecular ,biology ,Chemistry ,Chemiosmosis ,Protein Conformation ,Clinical Biochemistry ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,medicine.disease_cause ,Biochemistry ,Protein structure ,Bacterial Proteins ,Biophysics ,medicine ,Escherichia coli ,Inner membrane ,Efflux ,Multidrug Resistance-Associated Proteins ,Bacterial outer membrane ,Electrochemical gradient ,Molecular Biology ,Bacteria - Abstract
In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli, play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.
- Published
- 2015
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