Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

González-Mañas, Juan Manuel; Lakey, Jeremy H.; Pattus, F.

doi:10.1111/j.1432-1033.1993.tb17590.x

Cited by 22 publications

(13 citation statements)

References 28 publications

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“…The C-terminal domain of colicin A is composed of ␣-helices (40) and not ␤-sheets, as are outer membrane proteins (4,18). Furthermore, it contains a hydrophobic helical hairpin that allows it to insert spontaneously into both inner membranes and lipid bilayers (25,30,33,44). Such a hairpin could not constitute a signal for colicin A export to the outer membrane, as hydrophobic sequences have been shown to act as stop signals for protein export to the outer membrane (19,36).…”

Section: Discussionmentioning

confidence: 99%

Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

Cavard

2002

J Bacteriol

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Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.Colicins are proteins produced by strains of Escherichia coli carrying a colicinogenic plasmid and lethal for related E. coli strains. They are classified into two groups according to various characters. Colicins of group A are secreted, but those of group B are not. The mechanism of secretion of group A colicins is unknown. It involves one lipopeptide, called colicin lysis protein, which is expressed with the colicin. The colicin lysis proteins are thought to allow colicins to cross both the inner and outer membranes of E. coli and to release them in the medium late after synthesis (reviewed in references 14 and 46). Colicin A is a group A colicin. It is a pore-forming protein of 63 kDa, organized into three domains, like all colicins. It is produced in huge amounts during the SOS response along with the colicin A lysis protein, called Cal. Cal is synthesized as a precursor that is acylated and processed in the inner membrane before being exported to the outer membrane. Colicin A has been shown to accumulate in the cytoplasm before being released in the medium with the help of Cal at the end of induction (12).High-molecular-mass forms of colicin A have been detected in sodium dodecyl sulfate (SDS) gels loaded with unheated samples of producing cells (9, 31). They are poorly stained by Coomassie brilliant blue but can be visualized either on Western blots of the gels probed with antibodies to colicin A or on autoradiograms of gels loaded with cells labeled with radioactive amino acids. In this study, the heat-labile forms of colicin A were studied in induced ce...

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Section: Discussionmentioning

confidence: 99%

Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

Cavard

2002

J Bacteriol

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“…The corresponding lipids brominated at positions 9 -10 of the two acyl chains were also used, Br-DOPG and Br-DOPC (Avanti Polar Lipids, Alabaster, AL). Bromide addition was shown not to alter the physicochemical properties of DOPG and DOPC (25). Liposomes were prepared in a buffer containing 150 mM NaCl, 20 mM HEPES, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Conformational Changes Due to Membrane Binding and Channel Formation by Staphylococcal α-Toxin

Vécsey-Semjén

Lesieur

Möllby

et al. 1997

Journal of Biological Chemistry

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Conformational changes occurring upon membrane binding and subsequent insertion of staphylococcal ␣-toxin were studied using complementary spectroscopic techniques. Experimental conditions were established where binding could be uncoupled from membrane insertion but insertion and channel formation seemed to be concomitant. Binding led to changes in tertiary structure as witnessed by an increase in tryptophan fluorescence, a red shift of the tryptophan maximum emission wavelength, and a change in the near UV CD spectrum. In contrast to what was observed for the soluble form of the toxin, 78% of the tryptophan residues in the membrane-bound form were accessible to the hydrophilic quencher KI. At this stage, the tryptophan residues were not in the immediate vicinity of the lipid bilayer. Upon membrane insertion, a second conformational change occurred resulting in a dramatic drop of the near UV CD signal but an increase of the far UV signal. Tryptophan residues were no longer accessible to KI but could be quenched by brominated lipids. In the light of the available data on channel formation by ␣-toxin, our results suggest that the tryptophan residues might be dipping into the membrane in order to anchor the extramembranous part of the channel to the lipid bilayer.Despite the importance of membrane damaging proteins such as the immune proteins C9 of complement and perforin or pore-forming toxins from pathogenic bacteria, little is known about the precise mechanism that enables these proteins to convert from a water-soluble protein to a transmembrane channel. Channel-forming toxins such as staphylococcal ␣-toxin are extremely useful model proteins to address this issue since they are relatively easy to obtain in large amounts and will form channels in vitro.␣-Toxin is secreted by Staphylococcus aureus as a soluble monomeric protein. However, by the time it has reached its final goal, the toxin has turned into a heptameric transmembrane complex (for review see Refs. 1, 2). The various steps that allow this metamorphosis are the following. The toxin binds to an unidentified receptor on the surface of the target cell where it oligomerizes, thereby leading to a non-lytic pre-pore complex (3, 4). A second conformational change is required for insertion of the heptamer into the membrane in order to form the channel.Although no crystal structure is yet available for either the soluble or the heptameric form of the toxin, structural data based on mutagenesis, biochemical, and biophysical studies are rapidly accumulating. The soluble monomer is composed of at least two domains as illustrated by the fact that the protein unfolds in several steps upon acidification of the medium (5). It has been speculated that the N-and C-terminal halves of the polypeptide chain may constitute these domains based mainly on the observation that a central glycine-rich segment is very sensitive to proteases and most likely forms an interdomain loop (3, 6 -8). Amino acids implicated in membrane binding were found in both halves of the molecule (9 ...

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“…The synthesis of Br-DOPG, the vesicle preparations, and the fluorescence measurements were carried out as described previously (4,20,21).…”

Section: Methodsmentioning

confidence: 99%

Membrane Topology of the Colicin A Pore-forming Domain Analyzed by Disulfide Bond Engineering

Duché

Izard

González-Mañas

et al. 1996

Journal of Biological Chemistry

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Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected ␣-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the poreforming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the ␣-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the ␣-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that ␣-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.Colicin A is a bacteriocin that kills sensitive Escherichia coli cells by forming voltage-gated ion channels in cytoplasmic membranes. Like many toxins, colicin A is organized into structural domains, each of them carrying one function associated with the toxin's lethal activity (1). The N-terminal domain is involved in the translocation through the E. coli envelope, the central domain is responsible for the binding to a receptor on the bacterial surface, and the C-terminal domain possesses the pore-forming activity. The soluble form of this C-terminal domain obtained by mild proteolytic digestion was crystallized, and its three-dimensional structure was determined at 2.4 Å resolution. This molecule consists of a bundle of eight amphipathic ␣-helices surrounding two hydrophobic ␣-helices completely buried within the protein (2, 3).In vitro, the pore formation is divided into several steps including a voltage-independent membrane insertion and a voltage-dependent channel opening (4). The first step is initiated by an electrostatic interaction between colicin A and negatively charged phospholipids (5-7). Two models have been proposed to picture the conformational changes required for the colicin membrane insertion. In the first model, called the "umbrella model," the three layers of helices that form the soluble structure are rearranged so that the hydrophobic hairpin of helices 8 and 9 traverses the membrane, whereas the helical pair 1 and 2 folds out on the surface in the opposite direction to helices 4 -7 (8 -10). In the second model, called the "penknife model," the hydrophobic helical hairpin lies parallel to the membrane plane (4, 11, 12).Colicin channels inserted in planar lipid bilayers are opened by applying a trans-negative potential over a certain threshold voltage (13, 14). The voltage gating of colicins involves the translocation of parts of the protein across the membrane exposing different domains to the cis and trans solutions in the open and...

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Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

Cited by 22 publications

References 28 publications

Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

Conformational Changes Due to Membrane Binding and Channel Formation by Staphylococcal α-Toxin

Membrane Topology of the Colicin A Pore-forming Domain Analyzed by Disulfide Bond Engineering

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