Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.     

Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives

We have recorded the near- and far-ultraviolet circular dichroism spectra of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and derivatives of these toxins. The far-ultraviolet spectra of various forms of diphtheria toxin were virtually identical, implying that no major changes in secondary structure accompany proteolytic nicking or dimerization of toxin, or binding of the endogenous dinucleotide, adenylyl-(3'-5')-uridine 3'-monophosphate (AdoPUrdP). Alpha-helix content was estimated to be 29%, as compared with 8% for fragment A. Near-ultraviolet spectra were identical between nicked and intact diphtheria toxin. A broad negative transition with a minimum at 304 nm was assigned to the intrachain disulfide bridge within the B moiety. Dimeric diphtheria toxin showed perturbations of aromatic residues. Binding of AdoPUrdP to monomeric diphtheria toxin or of adenylyl-(3',5')-uridine (AdoPUrd) to fragment A perturbed one or more tryptophans. The latter results correlate with evidence for involvement of a tryptophan in NAD binding. Native exotoxin A was estimated to have 16% alpha-helix, and the activated form of exotoxin A, 11%. An enzymically active, 31 kDa proteolytic fragment of exotoxin A showed similar alpha-helix content (7%) to that of diphtheria toxin fragment A.     

Analysis of the structure-function relationship of Pseudomonas aeruginosa exotoxin A

Biochemical and genetic techniques have provided considerable insight into the structure-function relationship of one of the ADP-ribosyl transferases produced by Pseudomonas aeruginosa, exotoxin A. Exotoxin A contains a typical prokaryotic signal sequence which, in combination with the first 30 amino-terminal amino acids of the mature protein, is sufficient for exotoxin A secretion from P. aeruginosa. Determination of the nucleotide sequence and crystalline structure of this prokaryotic toxin allowed a molecular model to be constructed. The model reveals three structural domains of exotoxin A. Analysis of the identified domains shows that the amino-terminal domain (domain I) is involved in recognition of eukaryotic target cells. Furthermore, the central domain (domain II) is involved in secretion of exotoxin A into the periplasm of Escherichia coli. Evidence also implicates the role of domain II in translocation of exotoxin A from the eukaryotic vesicle which contains the toxin after it becomes internalized into susceptible eukaryotic cells via receptor-mediated endocytosis. The carboxy-terminal portion of exotoxin A (domain III) encodes the enzymatic activity of the molecule. The structure of this domain includes a cleft which is hypothesized to be the catalytic site of the enzyme. Several residues within domain III have been identified as having a direct role in catalysis, while others are hypothesized to play an important structural role.     

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.     

Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells.

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50% resistant and 50% sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CHO-K1 cells.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.

Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.     

Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.     

Requirement of the N-terminal region of orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.     

Identification of DNA restriction fragments of corynebacteriophage β corresponding to hypotoxic peptides of diphtheria toxin

Abstract An Xba I/ Eco RI restriction fragment (ca. 2000 bp) from corynebacteriophage β DNA was shown to contain the entire structural gene ( tox ) for diphtheria toxin, plus about 500 bp upstream from the amino terminus of the mature toxin. Restriction analysis and partial sequencing of this fragment permitted us to identify 3 large subfragments coding for hypotoxic peptides of diphtheria toxin. Two Mbo I restriction fragments, F1 (ca. 825 bp) and F3 (ca. 1000 bp), contained regions coding for the enzymatically active A fragment and most of the B fragment, respectively, of the toxin. An Msp I fragment, F2 (ca. 1450 bp), encoded a toxin peptide corresponding approximately to CRM45, a chain termination fragment lacking the carboxyl terminal region of the toxin. Fragments F1, F2, and F3 are permissible to clone in Escherichia coli under P1 + EK1 conditions according to current recombinant DNA guidelines.     

Prolylpeptide binding by the prokaryotic SH3-like domain of the diphtheria toxin repressor: a regulatory switch

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.     

Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.     

Complete nucleotide sequence and characterization of the 5'-flanking region of mammalian elongation factor 2 gene

The hamster elongation factor 2 gene was isolated from genomic libraries of diphtheria toxin- and Pseudomonas aeruginosa exotoxin A-resistant cells containing non-ADP-ribosylatable elongation factor 2, and its structure was determined by a combination of restriction endonuclease mapping and DNA sequence analysis. The entire gene is about 6 kilobases long and has 13 exons. Almost all the introns are about 90-200 bases long, except the first and third, which are about 1 kilobase and 400 bases long, respectively. The first exon is processed just after the initiation codon for translation. The promoter of this gene was also characterized. As this gene contains the mutation conferring resistance to diphtheria toxin and P. aeruginosa exotoxin A, introduction of this gene into mammalian cells results in expression of toxin resistance. Using this characteristic, gene expression by deletion mutants of the 5'-flanking region were examined, and results showed that about 60 base pairs upstream of the TATA sequence were most efficient for expression of the elongation factor 2 gene.     

Isolation and characterization of tox mutants of corynebacteriophage beta.

Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct form the structural gene for toxin, and that the CRM- strains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed.     

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.     

Entry of diphtheria toxin-protein A chimeras into cells

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.