Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine

Lukáč, Miloš; Collier, R. J.

doi:10.1021/bi00420a009

Cited by 36 publications

(17 citation statements)

References 15 publications

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“…1; Table 3). The results from this structure analysis support previous assignments of the residues that bind NAD (12)(13)(14). As proposed (15,31), H bonds and chargeneutralization interactions are formed between Arg-458 and the phosphate group of AMP.…”

Section: Resultssupporting

confidence: 87%

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

Dyda

Benhar

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Domain III of Pseudomonas aeruginosa exotoxin A catalyses the transfer of ADP-ribose from NAD to a modified histidine residue of elongation factor 2 in eukaryotic cells, thus inactivating elongation factor 2. This domain III is inactive in the intact toxin but is active in the isolated form.We report here the 2.5-A crystal structure of this isolated domain crystallized in the presence of NAD and compare it with the corresponding structure in the intact Pseudomonas aeruginosa exotoxin A. We observe a significant conformational difference in the active site region from Arg-458 to Asp-463. Contacts with part of domain II in the intact toxin prevent the adoption of the isolated domain conformation and provide a structural explanation for the observed inactivity. Additional electron density in the active site region corresponds to separate AMP and nicotinamide and indicates that the NAD has been hydrolyzed. The structure has been compared with the catalytic domain of the diphtheria toxin, which was crystallized with ApUp.Pseudomonas exotoxin A (PE) is a 66-kDa, 613-amino acid protein that is secreted by the bacterium Pseudomonas aeruginosa as one of its virulence factors (1, 2). The toxin binds to eukaryotic cells via the ubiquitous a2-macroglobulin receptor (3), and enters the cells by receptor-mediated endocytosis. Once in the cells, the toxin is cleaved by a specific protease and, after reduction of a disulfide bond (4), a C-terminal fragment (aa 280-613) is released and subsequently translocated to the cytosol, where it catalyzes the irreversible inactivation of eukaryotic elongation factor 2 (eEF-2) by ADP-ribosylation of a modified histidine (diphthamide) residue, leading to the arrest of protein synthesis and eventually to cell death (1, 5).The crystallographic determination of the PE structure has shown that PE is made up of three domains (6) and that there is a correspondence between the structural and the functional boundaries of the domains. Domain Ia (aa 1-252) is responsible for cell recognition. Removal of domain Ia or a specific point mutation at position 57 results in an inactive toxin due to its inability to bind to cells (7). Domain II (aa 253-364) is involved in the translocation of the toxin across intracellular membranes. The function of domain Ib (residues 365-399) has not been elucidated. Removal of part of domain lb (aa 365-380) results in a truncated but fully functional toxin (8). Domain III (aa 400-613) catalyzes the ADP-ribosylation of eEF-2 with the five C-terminal residues (REDLK) serving as an endoplasmic reticulum retention signal (9, 10). The functional boundary between domain lb and domain III is located at residue 400 (8).The ADP-transfer reaction catalyzed by domain III has been proposed to include formation of a binary complex of NAD and PE, binding of eEF-2 to the binary complex, and transfer of the ADP-ribose moiety of NAD to the diphthamide of eEF-2 as follows (11): PE NAD+ + eEF-2 ADP-ribose-eEF-2 + nicotinamide + H+.In the absence of eEF-2, a weak NAD-glycohydrolase a...

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Section: Resultssupporting

confidence: 87%

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

Dyda

Benhar

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…The important catalytic residues for ETA are Glu-553, His-440, Tyr-481, and Tyr-470, which were identified by structural and mutagenesis studies (9,13,34,35). The structure of the catalytic domain shows that the imidazole side chain of His-440, located at the base of the active site cleft, hydrogen bonds with the AMP ribose moiety of NAD ϩ and with the main chain carbonyl of Tyr-470 (13).…”

mentioning

confidence: 99%

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Armstrong

Yates

Merrill

2002

Journal of Biological Chemistry

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The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD ؉ but dependent upon pH. The lack of requirement for NAD ؉ to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model. The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity. The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues. The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states. Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2. These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.

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“…3A. The orbital stacking is of vital importance here and has been shown by mutagenesis studies of PAETA with Tyr470 Phe/Tyr481 Phe mutants still possessing enzymatic activity [81]. Aromatic ring stacking may explain the slightly different conformation that NAD possesses in the PAETA and diphtheria toxin structures compared with the structures of ADPRTs from the CT group [1,82].…”

Section: The Conserved Motifs That Characterize the Adprt Familymentioning

confidence: 99%

A family of killer toxins

Shone²,

2006

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Pathogenic bacteria are known to possess an arsenal of toxins and effectors that assist them in targeting and killing their host cells. The ADP-ribosylating toxins (ADPRTs) are a large family of dangerous and potentially lethal toxins. Examples of these toxins can be found in a diverse range of bacterial pathogens and they are the principal causative agents in many serious diseases including cholera, whooping cough and diphtheria. ADPRTs, as the name would suggest, break NAD into its component parts (nicotinamide and ADP-ribose) before selectively linking the ADP-ribose moiety to their protein target (Fig. 1). In the majority of these toxins, the targets are key regulators of cellular function and interference in their activity, caused by ADP-ribosylation, leads to serious deregulation of key cellular processes and in most cases, eventual cell death.This large family of toxins has been extensively studied with many structures of individual members determined. These include: diphtheria toxin (1TOX) The ADP-ribosylating toxins (ADPRTs) are a family of toxins that catalyse the hydrolysis of NAD and the transfer of the ADP-ribose moiety onto a target. This family includes many notorious killers, responsible for thousands of deaths annually including: cholera, enterotoxic Escherichia coli, whooping cough, diphtheria and a plethora of Clostridial binary toxins. Despite their notoriety as pathogens, the ADPRTs have been extensively used as cellular tools to study and elucidate the functions of the small GTPases that they target. There are four classes of ADPRTs and at least one structure representative of each of these classes has been determined. They all share a common fold and several motifs around the active site that collectively facilitate the binding and transfer of the ADP-ribose moiety of NAD to their protein targets. In this review, we present an overview of the physiology and cellular qualities of the bacterial ADPRTs and take an in-depth look at the structural motifs that differentiate the different classes of bacterial ADPRTs in relation to their function.

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Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine

Cited by 36 publications

References 15 publications

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

A family of killer toxins

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