Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

Abstract

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound Pseudomonas exotoxin A (PE) belongs to a family of bacterial nicotinamide adenine dinucleotide (NAD)-binding toxins that ADP ribosylate target proteins within eukaryotic cells (1).Crystal structures have been reported for four of these toxins: PE (2), diphtheria toxin (DT) (3), Escherichia coli heat-labile enterotoxin (LT) (4), and pertussis toxin (PT) (5), revealing a common fold for the catalytic ADP ribosylating domain, consisting of two approximately orthogonal, antiparallel (3 sheets flanked by several helices forming a large substrate binding cleft at the center of the domain. The folding topology is unlike the dinucleotide binding fold observed in several dehydrogenases (6).PE is a 613-amino acids protein that is composed of three structural domains. Domain 1 (1-252 and 365-399) binds to the ubiquitous a2-macroglobulin receptor of eukaryotic cells and initiates receptor-mediated endocytosis (7). In the cell, PE is cleaved at residue 280 within domain II (residues 253 to 364) by a specific protease (8); this cleavage activates the third domain. The portion of domain II (residues 280-364) remaining with the C-terminal fragment appears to translocate the fragment through intracellular membranes into the cytosol, where domain III (residues 400-613) acts by transferring the ADP ribose from NAD to a modified histidine in elongation factor-2 (EF-2). This irreversible covalent modification inhibits protein synthesis and leads to cell death. The function of domain lb (residues 365-399) is unknown and it can be entirely deleted without loss of toxin activity (9).In vitro, the intact toxin molecule has low affinity for NAD and exhibits no ADP ribosyl transferase activity (2). Treatment with reducing and denaturing agents is necessary to obtain an active molecule that can both bind to NAD and effect ADP ribosylation of EF-2 (10). The isolated domain III (PEIII) is active in the absence of this activation treatment (11). PEIII also exhibits a weak NAD glycohydrolase activity (12). We have determined (13) the structure of this catalytic domain complexed with the products of NAD hydrolysis, AMP and nicotinamide, thus effectively defining the active site. This structure revealed that the process of activation must involve the removal of helix 333 to 353 of domain TI from the vicinity of the active site loop 458 to 463 of domain ITT, thus permitting a conformational change in this loop that is necessary for NAD binding.In this structure, although the crystals were grown in the presence of NAD, resulting hydrolysis prevented the v...

NAD Binding Induces Conformational Changes in Rho ADP-ribosylating Clostridium botulinum C3 Exoenzyme

“…3E, yellow bond). A similar situation was observed in the crystal structure of Pseudomonas aeruginosa exotoxin A, a diphtheria-like toxin in complex with ␤-TAD, an NAD analog (PDB accession code 1AER) (15). In this structure, two molecules are present in the asymmetric unit with one that binds an intact ␤-TAD and the other that binds a ␤-TAD cleaved at this unexpected position.…”

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

“…It is proposed that initially a binary complex forms between NAD ϩ and the catalytic domain followed by the subsequent binding of eEF-2, representative of an ordered sequential reaction (11,12). In addition, stereochemical and kinetic data suggest that the reaction mechanism is likely an S N 1 nucleophilic substitution involving an oxocarbonium cation, despite the observed inversion of configuration for the glycosidic bond formed between the ribose and diphthamide (5,11,13,14). This inversion most likely results because the anomeric carbon of the nicotinamide ribose is susceptible to a backside nucleophilic attack from the N-1 of the imidazole portion of diphthamide (5).…”

“…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).…”

“…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). The nicotinamide ring stacks with Tyr-481 and Tyr-470 and is located near the nicotinamide where it is involved in Van der Waals interactions with the thiazole-ribose moiety of the nonhydrolyzable NAD ϩ analogue, ␤-TAD.…”

“…The nicotinamide ring stacks with Tyr-481 and Tyr-470 and is located near the nicotinamide where it is involved in Van der Waals interactions with the thiazole-ribose moiety of the nonhydrolyzable NAD ϩ analogue, ␤-TAD. Glu-553 forms a hydrogen bond with the oxygen of the thiazole-ribose of ␤-TAD (11,13) and may serve to cradle NAD ϩ in a reactive conformation, exposing the anomeric carbon for attack by the diphthamide of eEF-2 while still allowing the inversion of configuration at this carbon. Additionally, the negatively charged carboxylate of Glu-553 may stabilize a positively charged reaction intermediate (13).…”