Glycophorin as a receptor for Escherichia coli alpha-hemolysin in erythrocytes

Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes. However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles). Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes. Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin. With this purpose, a number of experiments were performed. (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes. (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis. (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts. (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA. (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity. The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC. The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.     

Locking the kink in the influenza hemagglutinin fusion domain structure

We have previously identified Trp(14) as a critical residue that stabilizes the kink in the boomerang structure of the influenza fusion domain and found that cells expressing hemagglutinin with a Trp(14) to Ala mutation cannot fuse with red blood cells. However, mutating another aromatic residue, Phe(9), on the other side of the kink did not have a significant effect on fusion or the ability of the mutant fusion peptide to bind to or perturb the bilayer structure of lipid model membranes. We reasoned that Phe is not as potent to contribute to the kink as the larger Trp and that the cooperation of Phe(9) and Ile(10) might be needed to elicit the same effect. Indeed, the double mutant F9A/I10A diminished cell-cell fusion and the ability of the fusion domain to bind to and perturb lipid bilayers in a similar fashion as the W14A mutant. A structure determination of F9A in lipid micelles by solution NMR shows that F9A adopts a similarly kinked structure as wild type. Distances between the two arms of the boomerang structure of wild type, F9A, W14A, and F9A/I10A in lipid bilayers were measured by double electron-electron resonance spectroscopy and showed that the kinks of W14A and F9A/I10A are more flexible than those of wild type and F9A. These results underscore the importance of large hydrophobic residues on both sides of the kink region of the influenza hemagglutinin fusion domain to fix the angle of the boomerang structure and thereby confer fusion function to this critical domain.     

Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity.

Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.     

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume,morphology, rheology and hemolysis

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis.We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry.Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%).The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis.For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure.HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca²⁺i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media.Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.     

Role of lipopolysaccharide on the structure and function of alpha-hemolysin from Escherichia coli

alpha-Hemolysin (HlyA) is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. Several studies suggested the relationship between HlyA and lipopolysaccharide (LPS). We have studied experimentally the role of LPS on the stability and function of this toxin. The HlyA conformation in both, LPS-free and LPS-bound forms was investigated by tryptophan fluorescence. Studies about HlyA thermal and chemical denaturation indicated that its stability increased in the presence of LPS. On the other hand, the presence of negative and polar residues on the LPS reduced the tendency of HlyA to self-aggregation, and they may be the reservoir of calcium, cation essential for the lytic action of this toxin on red blood cells. These results suggest that HlyA and LPS are combined mainly via hydrophobic force to form an active toxin which stability is favored by the LPS.     

Immunochemical evidence for the transmembrane orientation of glycophorin A. Localization of ferritin-antibody conjugates in intact cells.

Antibodies were raised in rabbits to a 51-amino acid cyanogen bromide-derived peptide of human erythrocyte glycophorin A which has been shown to represent the C-terminal end of the 131-residue polypeptide chain. Antibodies prepared by immunoadsorption were found to be directed against a chymotryptic-derived peptide (residues 102 to 118) of glycophorin A but were unreactive with either intact or proteolytically modified red blood cells. No cross-reactivity was observed with glycophorin B of human or sialoglycoproteins prepared from red blood cells of other mammalian species. Ferritin-antibody conjugates of such sera were applied to thin sections of intact red blood cells (frozen or protein embedded) and were found to localize exclusively to sites distributed uniformly along the inner surfaces of the membrane. No staining was seen on sections prepared from red blood cells from other species nor on sections of human red cells pretreated with unconjugated antisera. These results provide additional evidence in intact, fixed human erythrocytes that glycophorin A has a transmembrane orientation.     

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: Plausible implications in the toxic activity of the protein

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89^th position was replaced by aspartic acid and in the other both glycine and valine at the 88^th and 89^th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.     

Structural insight into the interaction between the p55 PDZ domain and glycophorin C

p55, a member of the membrane-associated guanylate kinase family, includes a PDZ domain that specifically interacts with the C-terminal region of glycophorin C in the ternary complex of p55, protein 4.1 and glycophorin C. Here we present the first NMR-derived complex structure of the p55 PDZ domain and the C-terminal peptide of glycophorin C, obtained by using a threonine to cysteine (T85C) mutant of the p55 PDZ domain and a phenylalanine to cysteine (F127C) mutant of the glycophorin C peptide. Our NMR results revealed that the two designed mutant molecules retain the specific interaction manner that exists between the wild type molecules and can facilitate the structure determination by NMR, due to the stable complex formation via an intermolecular disulfide bond. The complex structure provides insight into the specific interaction of the p55 PDZ domain with the two key residues, Ile128 and Tyr126, of glycophorin C.     

Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil alpha-helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two alpha-helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala366-Arg399 of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i). E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its alpha-helical content in both aqueous buffer and solvents that promote helix formation. (ii). Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a Kd of 43.6 +/- 4.9 nm or 2370 +/- 670 nm in the presence or absence of calcium, respectively. (iii). In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (Ki = 78.9 +/- 5.6 nm). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short alpha-helical flanking regions are sufficient for high affinity binding.     

A Mutant Influenza Virus That Uses an N1 Neuraminidase as the Receptor-Binding Protein

In the vast majority of influenza A viruses characterized to date, hemagglutinin (HA) is the receptor-binding and fusion protein, whereas neuraminidase (NA) is a receptor-cleaving protein that facilitates viral release but is expendable for entry. However, the NAs of some recent human H3N2 isolates have acquired receptor-binding activity via the mutation D151G, although these isolates also appear to retain the ability to bind receptors via HA. We report here the laboratory generation of a mutation (G147R) that enables an N1 NA to completely co-opt the receptor-binding function normally performed by HA. Viruses with this mutant NA grow to high titers even in the presence of extensive mutations to conserved residues in HA''s receptor-binding pocket. When the receptor-binding NA is paired with this binding-deficient HA, viral infectivity and red blood cell agglutination are blocked by NA inhibitors. Furthermore, virus-like particles expressing only the receptor-binding NA agglutinate red blood cells in an NA-dependent manner. Although the G147R NA receptor-binding mutant virus that we characterize is a laboratory creation, this same mutation is found in several natural clusters of H1N1 and H5N1 viruses. Our results demonstrate that, at least in tissue culture, influenza virus receptor-binding activity can be entirely shifted from HA to NA.     

Membrane insertion of Escherichia coli alpha-hemolysin is independent from membrane lysis

Escherichia coli alpha-hemolysin (HlyA) is a protein exotoxin that binds and lyses eukaryotic cell and model membranes in the presence of calcium. Previous studies have been able to distinguish between reversible toxin binding to the membrane and irreversible insertion into the lipid matrix. Membrane lysis occurs as the combined effect of protein insertion plus a transient perturbation of the membrane bilayer structure. In the past, insertion and bilayer perturbation have not been experimentally dissected. This has now been achieved by studying HlyA penetration into lipid monolayers at the air-water interface, in which three-dimensional effects (of the kind required to break down the bilayer permeability barrier) cannot occur. The study of native HlyA, together with the nonlytic precursor pro-HlyA, and of different mutants demonstrates that although some nonlytic variants (e.g. pro-HlyA) exhibit very low levels of insertion, others (e.g. the nonlytic mutant HlyA H859N) insert even more strongly than the lytic wild type. These results show that insertion does not necessarily lead to membrane lysis, i.e. that insertion and lysis are not "coupled" phenomena. Millimolar levels of Ca(2+), which are essential for the lytic activity, cause an extra degree of insertion but only in the case of the lytic forms of HlyA.     

A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, gammaENaC contained such a domain just distal to TM2 spanning Gln573-Pro600. Interactive domains were also localized within Met1-Gln54 and the last 17 residues of alpha- and betaENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of alphaENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 +/- 0.14 versus 2.5 +/- 0.80 nA of amiloridesensitive inward current at -80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 +/- 0.03 versus 0.67 +/- 0.07 for wild type. Mutant gammaENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573-Pro600, deletion of Gln573-Arg583 but not Thr584-Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573-Arg583 of gammaENaC are necessary for normal channel gating.     

Localization of the diphtheria toxin receptor-binding domain to the carboxyl-terminal Mr approximately 6000 region of the toxin

The carboxyl-terminal Mr = 5982 peptide of diphtheria toxin was prepared by specific cleavage of the toxin with hydroxylamine and purified by fast performance liquid chromatography. The identity of the peptide was established by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, reactivity with specific monoclonal antibodies, and amino-terminal sequence analysis. The Mr = 5982 peptide was shown to protect highly toxin-sensitive Vero cells from the lethal action of diphtheria toxin. This protection was shown to be due to inhibition of the initial step in the cytotoxic process, the binding of toxin to its receptor. These results strongly suggest that the Mr = 5982 carboxyl-terminal region (amino acid residues 482-535) is, or contains, the receptor-binding domain of diphtheria toxin.     

Cloning of the phycobilisome rod linker genes from the cyanobacterium Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.     

Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor

Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses.     

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.     

Structural analysis of the major human erythrocyte membrane sialoglycoprotein from Miltenberger class VII cells

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the red blood cells of an individual, homozygous for the Mi-VII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic peptide comprising the residues 40-61 of glycophorin A was deduced from manual sequence analyses. The Mi-VII-specific glycophorin A was shown to exhibit an arginine----threonine and a tyrosine----serine exchange at the positions 49 and 52 respectively. The threonine-49 residue was found to be glycosylated. Inhibition assays demonstrated that one of the Mi-VII-specific antigen determinants (Anek) is located within the residues 40-61 of glycophorin A and comprises sialic acid residue(s) attached to O-glycosidically linked oligosaccharide(s). Our data contribute to an understanding of the Miltenberger system and provide an explanation at the molecular level for the previous finding that the erythrocytes from the Mi-VII homozygote lack a high-frequency antigen (EnaKT), located within the residues 46-56 of normal glycophorin A.     

His-859 is an essential residue for the activity and pH dependence of Escherichia coli RTX toxin alpha-hemolysin

Escherichia coli alpha-hemolysin (HlyA) is a toxin protein that, in common with other members of the RTX family, contains a calcium-binding domain consisting of a number of Gly- and Asp-rich nonapeptides (17 in this case) repeated in tandem. Amino acid number 6 in these nonapeptides is almost invariably Asp, and occasionally Asn, but HlyA contains a His residue (number 859 in the chain) in position 6 of the last-but-one nonapeptide. HlyA mutants have been prepared, by site-directed mutagenesis, in which His-859 has been replaced by an Asn (H859N) or by Asp (H859D). HlyA exists in aqueous media in an aggregate-monomer equilibrium, but only the monomer containing bound Ca(2+) (HlyA.Ca) appears to be competent to achieve target membrane insertion and subsequent lysis. In mutant H859N, equilibrium appears to be shifted toward the aggregate, therefore the protein does not exchange Ca(2+) with the aqueous environment, no HlyA.Ca monomers are detected, and the protein lacks any membrane lytic activity. Mutant H859D in turn is almost indistinguishable from the wild-type regarding its calcium binding and membrane lytic activity, however, it differs significantly in its pH dependence. Wild-type HlyA activity decreases sigmoidally with pH, following rather closely the protonation curve of a His residue (apparent pK(a) approximately 6.5). With mutant H859D activity decreases almost linearly with pH and to a smaller extent. It can be concluded that His-859 plays a critical role in several aspects of HlyA activity, namely self-aggregation properties, calcium binding, hemolysis, and pH dependence.     

Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B

Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.     

Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli.

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.