Biomedical subjects

F Pattus

Publications and source records attributed to F Pattus.

At least 37 records · Page 2Linked to original sources

PubMed1995

Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD.

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen beta-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD:: omega background. The L2-deletion mutant only partially reconstituted super-susceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.

Amino Acid Sequence↗

PubMed1994

Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli.

A strain of Escherichia coli, selected on the basis of its resistance to colicin N, reveals distinct structural and functional alterations in unspecific OmpF porin. A single mutation [Gly-119-->Asp (G119D)] was identified in the internal loop L3 that contributes critically to the formation of the construction inside the lumen of the pore. X-ray structure analysis to a resolution of 3.0 A reveals a locally altered peptide backbone, with the side chain of residue Asp-119 protruding into the channel, causing the area of the constriction (7 x 11 A in the wild type) to be subdivided into two intercommunicating subcompartments of 3-4 A in diameter. The functional consequences of this structural modification consist of a reduction of the channel conductance by about one-third, of altered ion selectivity and voltage gating, and of a decrease of permeation rates of various sugars by factors of 2-12. The structural modification of the mutant protein affects neither the beta-barrel structure nor those regions of the molecule that are exposed at the cell surface. Considering the colicin resistance of the mutant, it is inferred that in vivo, colicin N traverses the outer membrane through the porin channel or that the dynamics of the exposed loops are affected in the mutant such that these may impede the binding of the toxin.

Amino Acid Sequence↗

PubMed1994

Uncoupled steps of the colicin A pore formation demonstrated by disulfide bond engineering.

Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

Amino Acid Sequence↗

PubMed1994

The cytolytic toxin aerolysin: from the soluble form to the transmembrane channel.

Aerolysin is a cytolytic toxin which forms channels in the plasma membranes of eucaryotic cells. The protein is secreted by Aeromonas hydrophila as an inactive protoxin. Its stability and water solubility are conferred by its ability to dimerize. Maturation of the protein occurs through proteolytic removal of a C-terminal peptide outside the secreting cell. Although the aerolysin which is so produced is still a dimer, it then has the ability to oligomerize. The oligomer is the active form of the toxin, capable of forming the transmembrane channels that disrupt cells. We review here the present knowledge about the structure of aerolysin in relation to the various steps in channel formation.

Aeromonas hydrophila↗

PubMed1994

All in the family: the toxic activity of pore-forming colicins.

Colicins are unusual bacterial toxins because they are directed against close relatives of the producing strain. They kill their targets in one of three distinct ways; via a ribonuclease or deoxyribonuclease activity or by forming pores in the target cell's membrane. This review deals with the steps involved in pore-forming colicin activity including, initial synthesis of the toxin, toxin release, receptor binding, translocation across the periplasm and pore formation in the cytoplasmic membrane. Special reference is made to the role of colicin in vivo, the structural changes occurring during pore formation and the role of the immunity protein.

Bacteria↗

PubMed1994

The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

Amino Acid Sequence↗

PubMed1994

Membrane-bound form of the pore-forming domain of colicin A. A neutron scattering study.

The ion-channel forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. This fragment forms a well-defined complex with dimyristoylphosphatidyl-glycerol which is thus amenable to neutron scattering studies. Neutron scattering experiments in the Guinier range (low angles) provided the mass and the stoichiometry of the complex (290,000 (+/- 10,000) M(r), 8.2 (+/- 0.5)), in fair agreement with previous determinations. By varying the neutron scattering length density of the solvent with 2H2O/H2O mixtures and therefore the contrast of the different components, the radial distribution of the protein and of the lipids was determined. Finally, an attempt was made to fit various models to the wider angle scattering data. This study suggests that the pore-forming fragment of colicin A lies mostly at the surface of the membrane, with the lipids arranged in a bilayer organization.

Colicins↗

PubMed1994

Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.

Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections. Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers. Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution. The protoxin (M(r) 52,000) adopts a novel protein fold. Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels.

Aeromonas↗

PubMed1993

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study.

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.

Chymotrypsin↗

PubMed1993

Dimerization stabilizes the pore-forming toxin aerolysin in solution.

Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.

Amino Acid Sequence↗

PubMed1993

Characterization of the receptor and translocator domains of colicin N.

Intact colicin N and various colicin derivatives, including a natural fragment lacking the first 36 amino-acid residues, a chymotryptic fragment lacking the first 66 amino acids and a thermolytic fragment comprising residues 183-387, were used to locate the regions involved in colicin-N uptake by sensitive Escherichia coli cells. Two separate domains of the molecule participate in colicin-N entry. Specific binding to OmpF receptor site requires a region located between residues 67-182. A N-terminal domain, located between residues 17-66, is involved during the translocation step after binding to receptor. Two sub-regions, residues 17-36 and residues 37-36, can be defined in this domain. The location and interactions between these domains are discussed in comparison to other colicins which use similar cell components for their uptake.

Binding, Competitive↗

PubMed1993

Fluorescence energy transfer distance measurements. The hydrophobic helical hairpin of colicin A in the membrane bound state.

The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.

Colicins↗

PubMed1993

Role of acidic lipids in the translocation and channel activity of colicins A and N in Escherichia coli cells.

Colicins A and N are pore-forming bacterial toxins that kill Escherichia coli cells. Their mode of action involves three steps; binding to specific receptors located in the outer membrane, translocation through this membrane and the periplasm, and channel formation in the inner membrane. In-vitro studies have shown that negatively charged phospholipids are an absolute requirement for the channel formation of colicin A. Using HDL11 strain, in which the phosphatidylglycerol (PtdGro) content was altered by varying the synthesis of the PtdGro-phosphate synthase, the effect of envelope PtdGro content on the activity of colicin A was studied in vivo. The formation by colicin A of a voltage-gated channel in the cytoplasmic membrane results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed by the toxin to cross the cell envelope. This lag time is higher when the cells have a reduced PtdGro level, suggesting that the receptor/translocation machinery of colicin A (OmpF, BtuB and Tol QRAB proteins) is altered in the absence of PtdGro. The rate of potassium efflux is also greatly reduced when the PtdGro content is decreased, suggesting that a certain level of PtdGro is indeed required for proper insertion of the colicin-A channel. In contrast, the activity of colicin N does not show any PtdGro dependence. The difference between the behavior of colicin A and that of colicin N is discussed.

Biological Transport↗

PubMed1993

Oligomerization of the channel-forming toxin aerolysin precedes insertion into lipid bilayers.

Oligomerization is a necessary step in channel formation by the bacterial toxin aerolysin. We have identified a region of aerolysin containing two tryptophans which influence the ability of the protein to oligomerize. Changing the tryptophan at position 371 or 373 to leucine resulted in mutant proteins that oligomerized at much lower concentrations than the wild-type toxin. Near-ultraviolet circular dichroism measurements showed that the tertiary structures of the L-371 and L-373 mutant toxins may be slightly different from the structure of wild type. Other single amino acid replacements in the same region of the protein as the two tryptophans appeared to have little or no effect on any properties of the protein. None of the changes we made had any measured effect on secretion of the protein by the bacteria. The L-373 and L-371 proteins induced chloride release from liposomes at lower concentrations than native toxin. Wild-type aerolysin solutions were completely unable to cause release when oligomeric toxin was absent or when it was removed by centrifugation. Aerolysin changed at H-132, which cannot form oligomers, was also inactive against liposomes. We conclude that aerolysin channels are produced by direct insertion of oligomers formed in solution, or assembled on the surface of the cell after binding to the receptor, and not by lateral diffusion of the monomer after it enters the lipid bilayer.

Amino Acid Sequence↗

PubMed1993

Interaction of the colicin-A pore-forming domain with negatively charged phospholipids.

The interaction of colicin-A thermolytic fragment with negatively charged liposomes was studied by fluorescence spectroscopy. 1,2-Dioleoyl-sn-glycero-3-phospho-1-sn-glycerol (Ole2GroPGro) containing liposomes do not significantly alter the fluorescence properties of the protein, and thus cannot give much information about this interaction. 1,2-Bis(9,10-dibromooleoyl-sn-glycero-3-phospho-1-sn-glycerol (Br4Ole2GroPGro) is easily synthesized by addition of bromine atoms to the double bond located at the mid-point of the fatty-acid acyl chain of Ole2GroPGro. The brominated phospholipid forms vesicles that strongly quench the protein fluorescence emission. The results presented here show that conversion of Ole2GroPGro to Br4Ole2GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allows for the estimation of the distribution of the quenching phospholipid molecules around the fluorophores [Yeager, M. D. & Feigenson G. W. (1990) Biochemistry 29, 4380-4392]. Binding of the protein to the vesicles is an irreversible process, since inserted molecules do not dissociate from the vesicle. From steady-state measurements, it can be concluded that in the membrane-bound form, the tryptophans are located within quenching distance of the bromine atoms, i.e. close to the lipid head-group/hydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid molecules in contact with the fluorescent domain of the protein is nine.

Bromine↗

PubMed1993

pH-dependent stability and membrane interaction of the pore-forming domain of colicin A.

Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A.

Calorimetry, Differential Scanning↗

PubMed1993

Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins.

The recently determined structures of three different protein toxins by X-ray crystallography has unexpectedly revealed a common membrane-insertion domain. This domain consists of an alpha-helical bundle of between seven and ten helices, some of which are hydrophobic. The three toxins, colicin, insecticidal delta-endotoxin and diphtheria toxin are directed towards different hosts, have different killing mechanisms and bear no sequence homology. The observation of a common membrane-insertion domain has implications for the design of therapeutic agents in combating disease.

Bacillus thuringiensis Toxins↗

PubMed1992

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.

Aeromonas hydrophila↗