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

Abstract

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...

A Catalytic Loop within Pseudomonas aeruginosa Exotoxin A Modulates Its Transferase Activity

“…Notably, there is sequence conservation at Asp 484 and Glu 486 within the Loop C region of these two diphthamide-specific toxins. Interestingly, Loop C did not appear in the original crystal structure of intact whole ETA (25); however, it was resolved in the structure of the catalytic domain of ETA (8,9). The three-dimensional structure of Loop N was not fully resolved in the catalytic domain structure that bound a less hydrolyzable NAD ϩ analog, ␤-methylene-thiazole-4-carboxamide adenine dinucleotide (␤-TAD ϩ ), as it was disordered (9), but was evident in the structures of whole ETA and the catalytic domain complexed with nicotinamide and AMP (8).…”

“…Only a few residues have been implicated to associate with eEF-2: His 426 , Tyr 481 , and His 440 ; however, these have not been confirmed. The crystal structures (8,9) show that His 440 is located at the base of the active site distant from the scissile N-glycosidic bond; therefore, contact with NAD ϩ is unlikely at this position. The three-dimensional structure shows Tyr 481 stacking against the nicotinamide ring of NAD ϩ (8,9).…”

“…The crystal structures (8,9) show that His 440 is located at the base of the active site distant from the scissile N-glycosidic bond; therefore, contact with NAD ϩ is unlikely at this position. The three-dimensional structure shows Tyr 481 stacking against the nicotinamide ring of NAD ϩ (8,9). Substitution of phenylalanine for Tyr 481 alters the rate of reaction, possibly by changing the orientation of the diphthamide on eEF-2 (10).…”

“…Therefore, loop regions were the first targets for investigating the inner workings of the active-site machinery of exotoxin A. In the crystal structures of the catalytic domain of ETA, two surface-exposed loops near the known active site were found (8,9). It was proposed that either or both of these loops act as clamps holding eEF-2 near the active site, or perhaps these loops work to stabilize the transition state structure during the ADP-ribosylation reaction.…”

“…This ADP-ribosylation reaction inactivates eEF-2 through the covalent attachment of the ADP-ribosyl moiety to the diphthamide residue of eEF-2, which results in cessation of protein synthesis and eventually cell death (6). Although the NAD ϩ substrate-docking site within ETA is well characterized (8,9), the eEF-2 substrate site is virtually unknown (10 -14). Only a few residues have been implicated to associate with eEF-2: His 426 , Tyr 481 , and His 440 ; however, these have not been confirmed.…”