22 results on '"Nikaido H"'
Search Results
2. Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives.
- Author
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Sjuts H, Vargiu AV, Kwasny SM, Nguyen ST, Kim HS, Ding X, Ornik AR, Ruggerone P, Bowlin TL, Nikaido H, Pos KM, and Opperman TJ
- Subjects
- Anti-Bacterial Agents chemistry, Binding Sites, Crystallography, X-Ray, Drug Discovery, Drug Resistance, Multiple, Bacterial drug effects, Escherichia coli drug effects, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Molecular Dynamics Simulation, Multidrug Resistance-Associated Proteins chemistry, Multidrug Resistance-Associated Proteins metabolism, Protein Structure, Tertiary, Pyrans chemistry, Pyrans pharmacology, Pyridines chemistry, Anti-Bacterial Agents pharmacology, Escherichia coli Proteins antagonists & inhibitors, Multidrug Resistance-Associated Proteins antagonists & inhibitors, Pyridines pharmacology
- Abstract
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
- 2016
- Full Text
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3. Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump.
- Author
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Kinana AD, Vargiu AV, May T, and Nikaido H
- Subjects
- Kinetics, Substrate Specificity, Escherichia coli Proteins metabolism, Multidrug Resistance-Associated Proteins metabolism, Naphthalenes metabolism
- Abstract
Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.
- Published
- 2016
- Full Text
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4. Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides.
- Author
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Bansal-Mutalik R and Nikaido H
- Subjects
- Acylation, Chromatography, Thin Layer, Electrophoresis, Polyacrylamide Gel, Fatty Acids analysis, Humans, Lipopolysaccharides metabolism, Micelles, Models, Biological, Mycobacterium smegmatis, Solutions, Cell Membrane metabolism, Lipid Bilayers metabolism, Mycobacterium metabolism, Phosphatidylinositols metabolism
- Abstract
Mycobacterium species, including the human pathogen Mycobacterium tuberculosis, are unique among Gram-positive bacteria in producing a complex cell wall that contains unusual lipids and functions as a permeability barrier. Lipids in the cell wall were hypothesized to form a bilayer or outer membrane that would prevent the entry of chemotherapeutic agents, but this could not be tested because of the difficulty in extracting only the cell-wall lipids. We used reverse micellar extraction to achieve this goal and carried out a quantitative analysis of both the cell wall and the inner membrane lipids of Mycobacterium smegmatis. We found that the outer leaflet of the outer membrane contains a similar number of hydrocarbon chains as the inner leaflet composed of mycolic acids covalently linked to cell-wall arabinogalactan, thus validating the outer membrane model. Furthermore, we found that preliminary extraction with reverse micelles permitted the subsequent complete extraction of inner membrane lipids with chloroform-methanol-water, revealing that one-half of hydrocarbon chains in this membrane are contributed by an unusual lipid, diacyl phosphatidylinositol dimannoside. The inner leaflet of this membrane likely is composed nearly entirely of this lipid. Because it contains four fatty acyl chains within a single molecule, it may produce a bilayer environment of unusually low fluidity and may slow the influx of drugs, contributing to the general drug resistance phenotype of mycobacteria.
- Published
- 2014
- Full Text
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5. Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels.
- Author
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Kojima S and Nikaido H
- Subjects
- Cytoplasm metabolism, Diffusion, Escherichia coli Proteins physiology, Penicillins pharmacokinetics, Porins physiology
- Abstract
Small, hydrophilic compounds such as β-lactams diffuse across the outer membrane of Gram-negative bacteria through porin channels, which were originally thought to be nonspecific channels devoid of any specificity. However, since the discovery of an ampicillin-binding site within the OmpF channel in 2002, much attention has been focused on the potential specificity of the channel, where the binding site was assumed either to facilitate or to retard the penetration of β-lactams. Since the earlier studies on porin permeability were done without the knowledge of the contribution of multidrug efflux pumps in the overall flux process across the cell envelope, in this study we have carefully studied both the porin permeability and active efflux of ampicillin and benzylpenicillin. We found that the influx occurs apparently by a spontaneous passive diffusion without any indication of specific binding within the concentration range relevant to the antibiotic action of these drugs, and that the higher permeability for ampicillin is totally as expected from the gross property of this drug as a zwitterionic compound. The active efflux by AcrAB was more effective for benzylpenicillin due to the stronger affinity and high degree of positive cooperativity. Our data now give a complete quantitative picture of the influx, efflux, and periplasmic degradation (catalyzed by AmpC β-lactamase) of these two compounds, and correlate closely with the susceptibility of Escherichia coli strains used here, thus validating not only our model but also the parameters obtained in this study.
- Published
- 2013
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6. Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations.
- Author
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Vargiu AV and Nikaido H
- Subjects
- Binding Sites, Drug Resistance, Multiple, Bacterial physiology, Escherichia coli drug effects, Escherichia coli metabolism, Escherichia coli Proteins antagonists & inhibitors, Ligands, Models, Molecular, Molecular Docking Simulation, Molecular Dynamics Simulation, Multidrug Resistance-Associated Proteins antagonists & inhibitors, Protein Binding, Protein Conformation, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Multidrug Resistance-Associated Proteins chemistry, Multidrug Resistance-Associated Proteins metabolism
- Abstract
Multidrug resistance in Gram-negative bacteria, to which multidrug efflux pumps such as the AcrB transporter makes a major contribution, is becoming a major public health problem. Unfortunately only a few compounds have been cocrystallized with AcrB, and thus computational approaches are essential in elucidating the interaction between diverse ligands and the pump protein. We used molecular dynamics simulation to examine the binding of nine substrates, two inhibitors, and two nonsubstrates to the distal binding pocket of AcrB, identified earlier by X-ray crystallography. This approach gave us more realistic views of the binding than the previously used docking approach, as the explicit water molecules contributed to the process and the flexible binding site was often seen to undergo large structural changes. We analyzed the interaction in detail in terms of the binding energy, hydrophobic surface-matching, and the residues involved in the process. We found that all substrates tested bound to the pocket, whereas the binding to this site was not preferred for the nonsubstrates. Interestingly, both inhibitors [Phe-Arg-β-naphthylamide and 1-(1-naphtylmethyl)-piperazine] tended to move out of the pocket at least partially, getting into contact with a glycine-rich loop that separates the distal pocket from the more proximal region of the protein and is thought to control the access of substrates to the distal pocket.
- Published
- 2012
- Full Text
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7. Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle extraction.
- Author
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Bansal-Mutalik R and Nikaido H
- Subjects
- Cardiolipins analysis, Cell Wall drug effects, Chromatography, Thin Layer, Corynebacterium glutamicum drug effects, Electrophoresis, Polyacrylamide Gel, Fatty Acids analysis, Lipids chemistry, Muramidase pharmacology, Solvents, Biochemistry methods, Cell Wall metabolism, Corynebacterium glutamicum metabolism, Lipids analysis, Lipids isolation & purification, Micelles
- Abstract
Cells of the Corynebacterium-Nocardia-Mycobacterium group of bacteria are surrounded by an outer membrane (OM) containing mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. This OM presumably acts as a permeability barrier that imparts high levels of intrinsic drug resistance to some members of this group, such as Mycobacterium tuberculosis, and its component lipids have been studied intensively in a qualitative manner over the years. However, the quantitative lipid composition of this membrane has remained obscure, mainly because of difficulties in isolating it without contamination from the inner cytoplasmic membrane. Here we use the extraction, with reverse surfactant micelles, of intact cells of Corynebacterium glutamicum and show that this method extracts the free OM lipids quantitatively with no contamination from lipids of the cytoplasmic membrane, such as phosphatidylglycerol. Although only small amounts of corynomycolate were esterified to arabinogalactan, a large amount of cardiolipin was present in a nonextractable form, tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex. Furthermore, we show that the OM contains just enough lipid hydrocarbons to produce a bilayer covering the cell surface, with its inner leaflet composed mainly of the aforementioned nonextractable cardiolipin and its outer leaflet composed of trehalose dimycolates, phosphatidylinositol mannosides, and highly apolar lipids, similar to the Minnikin model of 1982. The reverse micelle extraction method is also useful for extracting proteins associated with the OM, such as porins.
- Published
- 2011
- Full Text
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8. Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli.
- Author
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Takatsuka Y, Chen C, and Nikaido H
- Subjects
- Binding, Competitive, Cephalosporins chemistry, Cephalosporins pharmacology, Doxorubicin pharmacology, Drug Resistance, Multiple, Fluoresceins pharmacology, Ligands, Minocycline pharmacology, Models, Chemical, Models, Molecular, Mutation, Protein Binding, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Escherichia coli Proteins physiology, Multidrug Resistance-Associated Proteins metabolism, Multidrug Resistance-Associated Proteins physiology
- Abstract
The AcrB trimeric multidrug efflux transporter of Escherichia coli pumps out a very wide spectrum of compounds. Although minocycline and doxorubicin have been cocrystallized within the large binding pocket in the periplasmic domain of the binding protomer, nothing is known about the binding of many other ligands to this protein. We used computer docking to evaluate the interaction of about 30 compounds with the binding protomer and found that many of them are predicted to bind to a narrow groove at one end of the pocket whereas some others prefer to bind to a wide cave at the other end. Competition assays using nitrocefin efflux and covalent labeling of Phe615Cys mutant AcrB with fluorescein-5-maleimide showed that presumed groove-binders competed against each other, but cave-binders did not compete against groove-binders, although the number of compounds tested was limited. These results give us at least a hypothesis to be tested by more biochemical and genetic experiments in the future.
- Published
- 2010
- Full Text
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9. Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli.
- Author
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Nagano K and Nikaido H
- Subjects
- Cefamandole metabolism, Cefazolin metabolism, Cephaloridine metabolism, Cephalosporins metabolism, Cephalothin metabolism, Kinetics, Bacterial Outer Membrane Proteins metabolism, Escherichia coli Proteins metabolism, Lipoproteins metabolism, Membrane Transport Proteins metabolism, Multidrug Resistance-Associated Proteins metabolism, Multiprotein Complexes metabolism
- Abstract
Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.
- Published
- 2009
- Full Text
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10. How are the ABC transporters energized?
- Author
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Nikaido H
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Dimerization, Escherichia coli metabolism, Kinetics, Protein Subunits, ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters metabolism
- Published
- 2002
- Full Text
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11. Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli.
- Author
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Zgurskaya HI and Nikaido H
- Subjects
- Bacterial Proteins genetics, Biological Transport, Carrier Proteins genetics, Cytoplasmic Granules metabolism, Drug Resistance, Microbial, Escherichia coli genetics, Escherichia coli ultrastructure, Gene Expression Regulation, Bacterial, Hydrogen-Ion Concentration, Lipoproteins genetics, Magnesium metabolism, Membrane Proteins genetics, Membrane Transport Proteins, Multidrug Resistance-Associated Proteins, Protons, Bacterial Proteins metabolism, Carrier Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins, Lipoproteins metabolism, Membrane Proteins metabolism
- Abstract
AcrAB is a constitutively expressed, major multidrug efflux system of Escherichia coli. We have purified the cytoplasmic membrane component, AcrB, to near homogeneity, and reconstituted the protein into proteoliposomes. In the presence of DeltapH (outside acid), the protein catalyzed the extrusion of fluorescent phospholipids, which were then trapped by protein-free acceptor vesicles. Known substrates of AcrAB, such as bile acids, erythromycin, and cloxacillin, inhibited this activity. Addition of various drugs to AcrB-containing proteoliposomes, in the presence of DeltapH (inside acid) resulted in proton efflux, suggesting that AcrB is a proton antiporter. Interestingly, fluorescent lipid extrusion was accelerated strongly by the periplasmic protein AcrA in the presence of Mg2+, and at pH 5.0 AcrA alone produced a slow mixing of lipids of different vesicles, without causing the mixing of intravesicular material. These results suggest that AcrA brings two membranes together, and under certain conditions may even cause the fusion of at least the outer leaflets of the membranes, contributing to the ability of the AcrAB-TolC system to pump drugs out directly into the medium.
- Published
- 1999
- Full Text
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12. A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates.
- Author
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Liu J and Nikaido H
- Subjects
- Chromatography, High Pressure Liquid, Chromatography, Thin Layer, Fatty Acids chemistry, Fatty Acids isolation & purification, Fatty Acids metabolism, Kinetics, Magnetic Resonance Spectroscopy, Methylnitronitrosoguanidine pharmacology, Mutagenesis, Mycobacterium smegmatis drug effects, Mycolic Acids chemistry, Mycolic Acids isolation & purification, Novobiocin pharmacology, Spectrometry, Mass, Secondary Ion, Mycobacterium smegmatis genetics, Mycobacterium smegmatis metabolism, Mycolic Acids metabolism
- Abstract
Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a beta-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the alpha-branch of mycolic acids, followed by the joining of these two branches.
- Published
- 1999
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13. Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.
- Author
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Liu J, Rosenberg EY, and Nikaido H
- Subjects
- Calorimetry, Differential Scanning, Electron Spin Resonance Spectroscopy, Mycolic Acids chemistry, Temperature, Cell Wall chemistry, Lipids chemistry, Mycobacterium chelonae chemistry
- Abstract
The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. Hydrocarbon chains of much of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study, we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30 degrees C. Differential scanning calorimetry showed that much of the lipids underwent major thermal transitions between 30 degree C and 65 degrees C, that is at temperatures above the growth temperature, a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in the growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate labeled at different positions showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal, less fluid mycolic acid leaflet and an external, more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well-known intrinsic resistance of mycobacteria to such compounds.
- Published
- 1995
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14. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
- Author
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Davidson AL, Shuman HA, and Nikaido H
- Subjects
- Adenosine Triphosphate metabolism, Alleles, Carrier Proteins genetics, Cell Membrane metabolism, Escherichia coli genetics, Kinetics, Liposomes, Maltose-Binding Proteins, Models, Biological, Plasmids, Proteolipids isolation & purification, Proteolipids metabolism, Signal Transduction, ATP-Binding Cassette Transporters, Bacterial Proteins, Carrier Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins, Maltose metabolism, Monosaccharide Transport Proteins, Periplasmic Binding Proteins
- Abstract
Maltose transport across the cytoplasmic membrane of Escherichia coli is dependent on the presence of a periplasmic maltose-binding protein (MBP), the product of the malE gene. The products of the malF, malG, and malK genes form a membrane-associated complex that catalyzes the hydrolysis of ATP to provide energy for the transport event. Previously, mutants were isolated that had gained the ability to grow on maltose in the absence of MBP. After reconstitution of the transport complex into proteoliposomes, measurement of the ATPase activity of wild-type and mutant complexes in the presence and absence of MBP revealed that the wild-type complex hydrolyzed ATP rapidly only when MBP and maltose were both present. In contrast, the mutant complexes have gained the ability to hydrolyze ATP in the absence of maltose and MBP. The basal rate of hydrolysis by the different mutant complexes was directly proportional to the growth rate of that strain on maltose, a result indicating that the constitutive ATP hydrolysis and presumably the resultant cyclic conformational changes of the complex produce maltose transport in the absence of MBP. These results also suggest that ATP hydrolysis is not directly coupled to ligand transport even in wild-type cells and that one important function of MBP is to transmit a transmembrane signal, through the membrane-spanning MalF and MalG proteins, to the MalK protein on the other side of the membrane, so that ATP hydrolysis can occur.
- Published
- 1992
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15. In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.
- Author
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Sen K and Nikaido H
- Subjects
- Bacterial Outer Membrane Proteins biosynthesis, Bacterial Outer Membrane Proteins isolation & purification, Electrophoresis, Polyacrylamide Gel, Ion Channels metabolism, Macromolecular Substances, Methionine metabolism, Molecular Weight, Porins, Spheroplasts metabolism, Bacterial Outer Membrane Proteins metabolism, Escherichia coli metabolism
- Abstract
It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein.
- Published
- 1990
- Full Text
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16. Specificity of diffusion channels produced by lambda phage receptor protein of Escherichia coli.
- Author
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Luckey M and Nikaido H
- Subjects
- Bacteriophage lambda, Carbohydrate Metabolism, Diffusion, Receptors, Virus isolation & purification, Escherichia coli physiology, Ion Channels physiology, Receptors, Virus physiology
- Abstract
The lamB protein, the receptor for phage lambda, was purified from the outer membrane of Escherichia coli K-12 by extraction with Triton X-100 and EDTA, chromatography on DEAE-Sephacel in Triton X-100, exchange of Triton for cholate by gel filtration, and chromatography on Sephacryl S-200 in cholate, NaCl, and EDTA. The purified protein appeared to exist as several oligomeric species. In an equilibrium retention assay with reconstituted vesicles containing phospholipids and lipopolysaccharide, the lamB protein conferred permeability for disaccharides. In a liposome swelling assay designed to measure rates of diffusion, the lamB protein conferred permeability to phospholipid liposomes for a variety of substrates. The rates obtained indicate the permeation facilitated by the lamB protein is specific, discriminating among substrates by both size and configuration. For example, maltose diffused into liposomes 40 times faster than sucrose, about 8 times faster than cellobiose, and about 12 times faster than maltoheptaose. The results suggest that the lamB protein forms a transmembrane channel containing a site (or sites) that loosely interacts with the solutes.
- Published
- 1980
- Full Text
- View/download PDF
17. Amino acid sequence homology among the major outer membrane proteins of Escherichia coli.
- Author
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Nikaido H and Wu HC
- Subjects
- Amino Acid Sequence, Bacterial Outer Membrane Proteins, Base Sequence, DNA, Bacterial genetics, Genes, Genes, Bacterial, Plasmids, Structure-Activity Relationship, Bacterial Proteins genetics, Escherichia coli genetics, Membrane Proteins genetics
- Abstract
Analysis of amino acid sequences reported for the major outer membrane proteins of Escherichia coli, including the porins (OmpF, OmpC, and PhoE), the phage lambda receptor (LamB), and another protein (OmpA), revealed several regions of local homology that is statistically significant. The implications of this observation are discussed in relation to the evolutionary origins of these proteins, as well as to the mechanism of export of these proteins to the outer membrane.
- Published
- 1984
- Full Text
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18. Maltose transport in membrane vesicles of Escherichia coli is linked to ATP hydrolysis.
- Author
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Dean DA, Davidson AL, and Nikaido H
- Subjects
- Biological Transport, Cell Membrane metabolism, Escherichia coli genetics, Hydrolysis, Kinetics, Proton-Translocating ATPases metabolism, Species Specificity, Adenosine Triphosphate metabolism, Escherichia coli metabolism, Maltose metabolism
- Abstract
We examined the energy requirement for maltose transport in right-side-out membrane vesicles derived from Escherichia coli. When membrane vesicles were made from strains producing tethered maltose-binding proteins by dilution of spheroplasts into phosphate buffer, those from an F0F1 ATPase-containing (unc+) strain transported maltose in the presence of an exogenous electron donor, such as ascorbate/phenazine methosulfate, at a rate of 1-5 nmol/min per mg of protein, whereas those from an isogenic unc- strain failed to transport maltose. Transport in vesicles obtained from the latter strain could be restored in the presence of electron donors if the vesicles were made to contain NAD+ and either ATP or an ATP-regenerating system. ATP hydrolysis was apparently required for transport, since nonhydrolyzable ATP analogues did not sustain transport. Maltose transport significantly increased ATP hydrolysis in ATP-containing vesicles from unc- cells. Finally, ATP-containing vesicles from unc- strains producing normal maltose-binding proteins could accumulate maltose in the absence of electron donors. These results provide convincing evidence that it is the hydrolysis of ATP that drives maltose transport, and probably also other periplasmic-binding-protein-dependent transport systems.
- Published
- 1989
- Full Text
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19. Extended deletions in the histidine-rough-B region of the Salmonella chromosome.
- Author
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Nikaido H, Levinthal M, Nikaido K, and Nakane K
- Subjects
- Alcohol Oxidoreductases metabolism, Chromatography, Chromosome Mapping, Isomerases metabolism, Ligases metabolism, Molecular Biology, Phosphotransferases metabolism, Salmonella cytology, Salmonella enzymology, Chromosomes, Genes, Histidine biosynthesis, Mutation, Nucleotidyltransferases metabolism, Salmonella metabolism
- Published
- 1967
- Full Text
- View/download PDF
20. Studies on the biosynthesis of cell wall polysaccharide in mutant strains of Salmonella. II.
- Author
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NIKAIDO H
- Subjects
- Cell Wall, Polysaccharides, Salmonella metabolism
- Published
- 1962
- Full Text
- View/download PDF
21. SEMIROUGH STRAINS OF SALMONELLA.
- Author
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NAIDE Y, NIKAIDO H, MAEKELAE PH, WILKINSON RG, and STOCKER BA
- Subjects
- Biochemical Phenomena, Biochemistry, Carbohydrate Metabolism, Chromatography, Complement Fixation Tests, Mutation, Polysaccharides, Polysaccharides, Bacterial, Research, Salmonella
- Published
- 1965
- Full Text
- View/download PDF
22. Studies on the biosynthesis of cell-wall polysaccharide in mutant strains of Salmonella. I.
- Author
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NIKAIDO H
- Subjects
- Carbohydrate Metabolism, Cell Wall, Polysaccharides metabolism, Salmonella metabolism
- Published
- 1962
- Full Text
- View/download PDF
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