Localization of the active site of diphtheria toxin

Zhao, Jian; London, Erwin

doi:10.1021/bi00409a041

Cited by 32 publications

(21 citation statements)

References 51 publications

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“…This is consistent with recent crystallographic studies of Choe et al (6), who found that residues 32 to 54 are included within the CL2 loop of DT, which appears to cover the active-site region. Amino acid modification studies also support the idea of a close or direct association of residues in this sequence with the active site of DT, finding that alteration of Lys-39 (19) and Gly-52 (9) affects enzyme activity.…”

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confidence: 55%

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Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity

Olson

1993

J Bacteriol

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Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A have the same molecular mechanism of toxicity; both toxins ADP-ribosylate a modified histidine residue in elongation factor 2. To help identify amino acids involved in this reaction, sequences in DT that share homology with P. aeruginosa exotoxin A were synthesized and examined for a role in the ADP-ribosyltransferase reaction. By using this approach, residues 32 to 54 of DT were found to define an epitope associated with antibody-mediated inhibition of DT enzyme activity. This lends further support to the notion that residues in this region of DT are involved in the enzymatic reaction.Most clinical symptoms of diphtheria are attributed to a toxin produced by pathogenic strains of Corynebacterium diphtheriae. This diphtheria toxin (DT) is one of many bacterial toxins whose molecular mechanism of action involves an ADP-ribosyltransferase activity (7). Other toxins having this activity, such as cholera toxin, pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas exoenzyme S, and Clostridium C2, C3, and iota toxins, exert different effects on cellular functions, and these differences reflect their substrate specificities.Structure-function analyses of functionally related enzymes have, in many instances, led to the identification of functional sequence motifs. Although common sequence patterns can be identified in the enzymatically active region of ADP-ribosylating toxins having identical or closely related substrates, these same patterns are not apparent in ADP-ribosylating toxins having more distantly related substrates. An inability to identify a common sequence motif in the ADP-ribosylating toxins has precluded definitive identification of amino acids directly involved in this reaction.Many approaches have been used to help confirm the identity of key functional residues in ADP-ribosylating toxins. Crystallographic studies of Pseudomonas exotoxin A (PE) (1), DT (6), and E. coli heat-labile toxin (16) have localized the active-site region of these toxins. Numerous amino acid alteration and site-directed mutagenesis studies have also led to the identification specific residues within ADP-ribosylating toxins that affect enzyme activity. Studies described in this report used synthetic peptides to help confirm the identities of residues in DT that are associated with the ADP-ribosyltransferase reaction. By using this approach, a region between residues 32 and 54 of DT was found to define an epitope that affects DT enzymatic activity.Selection and immunological evaluation of DT synthetic peptides. Although DT and PE maintain the same molecular mechanism of action and a common substrate (7, 11), only limited sequence similarity between the two toxins has been identified (2, 4, 10, 19). The amino acid sequences within the enzymatically active region of DT and PE have been aligned in Fig. 1 to maximize recognition of sequence similarities. Since common sequences are likely candidates for common 898 enzymatic functions, sequences in DT that are conser...

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confidence: 55%

“…Although DT and PE maintain the same molecular mechanism of action and a common substrate (7,11), only limited sequence similarity between the two toxins has been identified (2,4,10,19). The amino acid sequences within the enzymatically active region of DT and PE have been aligned in Fig.…”

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confidence: 99%

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity

Olson

1993

J Bacteriol

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“…1A) demonstrates that the disappearance of the 20-kDa peptide correlates with the appearance of a new band at -54 kDa, which represents the 20-kDa fragment of DTA attached to DTB by the cystine bridge (Cys-186-Cys-201). Amino acid sequencing revealed that the 20-kDa fragment is the DTA fragment created by trypsin removal of 39 residues from the amino terminus (23). Because the 20-kDa fragment does not express nuclease activity in this assay, it appears that some or all of the amino terminus is required for nuclease activity.…”

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confidence: 88%

Localization of diphtheria toxin nuclease activity to fragment A

et al. 1992

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). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.Diphtheria toxin (DTx) is synthesized as a single polypeptide chain that can be "nicked" by limited proteolysis to generate A and B subunits (DTA and DTB, respectively) that remain connected by a cystine bridge (7,14,16). DTB is involved in receptor binding; following sequestration in endosomes and subsequent acidification of the endosomal compartment, DTA is transported to the cytosol (8,14,22). DTA is involved in the ADP ribosylation of elongation factor 2, which leads to the inhibition of protein synthesis (7,14,16). We and others have noted that extensive toxin-induced translation inhibition is not correlated with cell death in human K562 cells (4,19); these cells are lysis resistant to doses of DTx as high as 7.5 ,ug/ml despite the rapid and complete abrogation of protein synthesis (4). In addition, extensive inhibition of protein synthesis by unrelated treatments does not lead to the prelytic intemucleosomal DNA cleavage that we have observed in DTx-intoxicated cells or to cell lysis (4). These and other findings (5) led us to propose that DTx triggers the programmed cell death pathway (4). Testing this hypothesis resulted in the discovery of a nuclease activity intrinsic to the DTx molecule (3). Detection of this activity is highly reproducible (10), optimal reaction conditions have been established (13), and an ADP ribosyltransferase (ADPrT)-defective form of DTx (called CRM197) exhibits greater nuclease activity than DTx itself (2). Moreover, nuclease activity was found to comigrate with the DTA portions of both DTx and CRM197 during electrophoresis in DNA-containing sodium dodecyl sulfate (SDS) gels (2, 3, 10) and with whole DTx and CRM197 during native gel electrophoresis (2), even though each of these forms of DTx migrates with a distinctive mobility in each gel system. We now report that the nuclease activity exhibited by DTx is intrinsic to DTA. chromatography of DTx and trypsin-generated DTA (21); therefore, they proposed that the nuclease activity of their DTx preparations resides in an uncharacterized contaminating protein. Moreover, they showed that trypsin cleavage of intact DTx results in a severe decrease in total observed nuclease activity and a simultaneous increase in ADPrT activity (21). In contrast, we observed that endoproteinase ArgC-cleaved DTx remains fully active and that the amount of DTx-associated nuclease activity corresponds to the amount of DTA generated ...

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“…This active-site loop may be in a different conformation in nucleotide-free DT, in which these interactions are absent. The proposal that the loop undergoes a conformational change upon nucleotide binding is supported by a study that found Lys 39 changes its accessibility to proteases upon NAD or ApUp binding (Zhao & London, 1988). The importance of the loop in nucleotide binding is also suggested by the existence of a mutant DT, known as CRM 197, which has Glu substituted for Gly at position 52 and is unable to bind NAD (Lory et a]., 1980).…”

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Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections ( F > l o ( F ) ) , yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp.The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.Keywords: ADP-ribosyltransferase; diphtheria; membrane insertion Diphtheria toxin (DT) is secreted from toxic strains of the bacterium Corynebacterium diphtheriae. Toxigenic conversion of C. diphtheriae occurs by lysogenization with corynephage 0 (Freeman, 1951), which carries the DT gene encoding a 535-residue protein (M, 58,342) (Uchida et al., 1971; Greenfield et al., 1983). DT kills eukaryotic cells by inactivating an essential component of the protein translation machinery, elongation factor 2 (EF-2) (Collier, 1975).DT is produced as a protomer of 2 fragments connected by a loop containing a proteolysis site and a disulfide bond. Limited proteolysis with trypsin and reduction of the disulfide separates the 2 fragments, which are denoted fragment A and fragment B (Drazin et al., 1971). Fragment A consists of an independent folding domain, the catalytic (C) domain (residues 1-190) (Choe et al., 1992). Fragment B consists of 2 folding domains, the transmembrane (T) domain (residues 191-378) and receptor-binding (R) domain (residues 379-535) (Choe et al., 1992).

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Localization of the active site of diphtheria toxin

Cited by 32 publications

References 51 publications

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity

Localization of diphtheria toxin nuclease activity to fragment A

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

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